EPIPHYTOTIC AND CONTROL OF
ASCOCHYTA BLIGHT OF CHICKPEA CAUSED
BYAscochyta rabiei (Pass.) Labr.
IN ERBIL PROVINCE
A THESIS
SUBMITTED TO THE COLLEGE OF AGRICULTURE
UNIVERSITY OF SALAHADDIN-ERBIL
IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE
MASTER DEGREE IN AGRICULTURE
PLANT PROTECTION
(Plant Pathology)
By QASIM ABDULLAH OMER MARZANI
B.Sc. Agriculture (plant protection), Baghdad University, 1990
January - 2003
Rebandan - 2702
SUMMARY
Ascochyta blight of chickpea (Cicer arietinum L.) caused by Ascochyta
rabiei(Pass.) Labr. has been recorded for the first time in Erbil province . The
results of field surveys conducted in five locations in the province during
March, April, and May of 2002, showed that the disease exists in all these
locations. The disease incidence ranged between 40-100%, while disease
score range was 0.45-5.00 using 0-5 scale and some fields were completely
devastated and not harvested. The presence of chickpea debris of the previous
season and sowing infested seeds are mainly the reasons behind increasing of
disease incidence in the fields that were sown in early spring.
The pathogen A. rabiei was isolated from chickpea plants, debris and
seeds. From the pathogenicity tests of the pathogen, we found a typical
symptoms on the green plant parts of both desi and kabuli chickpea varieties.
On the basis of pathogenicity on 5 selected cultivars, three pathotypes were
determined from 13 isolates of A. rabiei which collected from chickpea
growing areas in Erbil province, and the highly virulent one was pathotype C.
The host range tests of A. rabiei revealed that in addition to its ability to
infect the genus Cicer, seven other legume species were also infected by the
pathogen. Simple disease symptoms with no formation of pycnidia, appeared
on these plant species, further, the pathogen was re-isolated from these hosts.
The perfect stage(teleomorph) of A. rabiei was recorded in Erbil province
which indicates the presence of both compatible mating types, mating type I
and mating type II, in the region.
Ascochyta blight appeared and developed on chickpea plant when
weekly average of maximum temperature, relative humidity, rainfall, and leaf
wetness period were 10.29oC, 70.07%, 47.2 mm and 16h, respectively.
The seed yield quantity and quality decreased with the increase of
disease severity. The results of planting seven cultivars showed that the
Winter sowing
was suitable with resistant cultivars. Shifting sowing date from winter to early
Spring, decreased the disease variables, but the yield components were also
decreased.
Screening
of
seven
chickpea
cultivers
revealed
that
local
landrace(Harir), Dijla, Ghab-2 and Ghab-3, were resistant cultivars. Seed
treatment with Raxil, Dividened, and Benlate were most effective in
protecting chickpea seedlings from infection. The results of foliar application
in the field showed ineffective of spray fungicides during rainy seasons and
with the susceptible cultivars, while in greenhouse tests, the most effective
fungicides were Raxil and Topas.
Acknowledgements
Firstly, I would like to thank the higher education council of Kurdistan region
and the Presidency of Salahaddin University, Erbil for opportunity they have offered
to me to make such a new study on Ascochyta blight of chickpea. Many thanks also to
Deanery of the college of Agriculture especially Dr. Farhad H. Aziz, the Dean, and the
head of Plant Protection Department, Mr. Yaqoub I. Elia, for their cooperation. I
would especially like to thank my supervisor, Mr. Yaqoub I. Elia, for his advice,
encouragement and guidance throughout the period of this study. I would to thank
the College of Science, Department of Biology, for allowing me to achieve parts of my
scientific research in the greenhouses. Special thanks to Mr. Yasin for his serious
cooperation and providing facilities. Thanks to Erbil agricultural research center for
their helps by providing me the certified seeds of some chickpea cultivars and other
legumes.
Very special thanks and gratitude to Dr. Muzahim A. Abdullah for his
advising and encouraging me during the study, besides his great help for offering the
references. I also wish to thank Mr. Adel Omran who took time to review all chapters
and made constructive suggestions throughout the period of writing this thesis. My
deepest thanks to Dr. Kahlid H. Taha, Dr. Akram O. Ismail and Mr. Wazeer A.
Hassan for their guidance and assistance during this study.
My Special thanks to Dr. Muehlbauer (from Washington State UniversityUSA), Dr. G. Kahl(Frankfurt University-Germany) and Dr. T. Bretag(Victorian
Institute for dryland Agriculture-Australia) for their supports by sending the recent
papers about the study by the Air-mail and E-mail. I am most indebted to my
colleague Mr. Abdulrahim O. Mustafa for his continuous supports and facilities,
starting from the beginning of the study until the date of final examination.
I truly indebted to a large number of individuals who assisted me, these include Mr.
Nawzad, Mr. Sardar, Mr. Omed, Mr. Harith, Mr. Sadraddin, Mr. Matti(in FAOlibrary), for their supports and to all staff members in Department of Plant
Protection. Finally, I apologize to all that assisted me in my work and I may forget to
mention their names in this regard.
Contents
Contents
A THESIS .................................................................................................................................................. 1
SUMMARY .................................................................................................................................................. 2
ACKNOWLEDGEMENTS ........................................................................................................................ 4
CONTENTS ................................................................................................................................................ I
CHAPTER 1 INTRODUCTION ................................................................................................................ 1
INTRODUCTION....................................................................................................................................... 1
CHAPTER 2 LETIRATURE REVIEW ..................................................................................................... 4
2.1 CHICKPEA DISEASES ............................................................................................................................... 4
2.2 GEOGRAPHICAL DISTRIBUTION OF ASCOCHYTA BLIGHT DISEASE.......................................................... 4
2.3 THE ECONOMIC IMPORTANCE OF THE DISEASE ...................................................................................... 5
2.4 DISEASE SYMPTOMS AND DEVELOPMENT............................................................................................... 6
2.5 THE CAUSAL ORGANISM .......................................................................................................................... 9
2.5.1 Identification of the pathogen ..................................................................................................... 9
2.5.2 The sexual stage ........................................................................................................................ 10
2.5.3 Physiologic races, pathotypes and isolates of A. rabei ........................................................ 12
2.6 EPIPHYTOLOGY OF THE DISEASE .......................................................................................................... 14
2.7 SOURCES OF INFECTION ....................................................................................................................... 16
2.7.1 Infected seeds ............................................................................................................................ 16
2.7.2 Infected chickpea debris ........................................................................................................... 17
2.7.3 Host range (alternative hosts) .................................................................................................. 18
2.8 CONTROL METHODS.............................................................................................................................. 20
2.8.1 Agricultural methods .................................................................................................................. 20
2.8.2 Control by planting resistant cultivars ..................................................................................... 23
2.8.3 Chemical control......................................................................................................................... 26
CHAPTER 3 MATERIALS ANDMETHODS ......................................................................................... 31
3.1 MATERIALS ............................................................................................................................................ 31
3.1.1 Instruments and equipment ...................................................................................................... 31
3.1.2 Culture media ............................................................................................................................. 32
3.2 METHODS .............................................................................................................................................. 32
i
3.2.1 Field survey ................................................................................................................................. 32
3.2.2 Isolation and identification of the pathogen ............................................................................ 35
3.2.3 Pathogenicity tests ..................................................................................................................... 37
3.2.4 Study of virulence of A. rabiei isolates .................................................................................... 38
3.2.5 Study of the host range ............................................................................................................. 39
3.2.6 Production of sexual stage (Pseudothcia) in the laboratory ................................................ 39
3.2.7 Effect of natural environmental factors on the disease ........................................................ 42
3.2.8 Effect of the disease on yield quantity and quality ................................................................ 43
3.2.9 Control Methods ......................................................................................................................... 44
CHAPTER 4 RESULTS AND DISCUSSIONS ..................................................................................... 53
4.1 FIELD SURVEY ....................................................................................................................................... 53
4.2 DISEASE SYMPTOMS IN THE FIELD ........................................................................................................ 56
4.3 ISOLATION AND IDENTIFICATION OF A. RABIEI ...................................................................................... 58
4.3.1 From infected plants .................................................................................................................. 58
4.3.2 From chickpea debris ................................................................................................................ 60
4.3.3 From chickpea seeds ................................................................................................................ 60
4.4 PATHOGENICITY TESTS ......................................................................................................................... 60
4.5 STUDY OF VIRULENCE OF A. RABIEI ISOLATES ..................................................................................... 61
4.6 THE HOST RANGE .................................................................................................................................. 63
4.6.1 Fitness costs ............................................................................................................................... 63
4.7 PRODUCTION OF SEXUAL STAGE .......................................................................................................... 65
4.7.1 On naturally infected chickpea debris ..................................................................................... 65
4.7.2 On artificially inoculated chickpea debris ............................................................................... 68
4.8 STUDY OF THE EFFECT OF ENVIRONMENTAL FACTORS ON THE DISEASE ............................................ 69
4.9 EFFECT OF THE DISEASE ON SEED YIELD QUANTITY AND QUALITY ...................................................... 72
4.10 CONTROL METHODS ........................................................................................................................... 72
4.10.1 Effect of sowing date on the ................................................................................................... 72
4.10.2 Screening Chickpea germplasm ............................................................................................ 82
4.10.3 Chemical control ...................................................................................................................... 86
REFERENCES ........................................................................................................................................ 97
ii
MSc. Thesis – Qasim Marzani
Chapter 1. Introduction
Chapter 1 Introduction
INTRODUCTION
Chickpea (Cicer arietinum L.) is an important legume crop of dryland
agriculture throughout West Asia, around the Mediterranean Sea, in parts of
East Africa, and Latin America (Reddy,1984a). It is the third most important
pulse crop in the world, while it ranks first on the Indian subcontinent and in
the Mediterranean basin (Anonymous, 1994). It is believed that the origin of
chickpea is South-east Turkey and Syria (Vander Macsen, 1987). Two types
of chickpea, generally erect and bushy, are grown in the world: desi [with
small, angular, and colored seed] and kabuli[with large, ram-head-shaped, and
beige-colored seed](Reddy and Singh,1990a).
Chickpea is spring-sown crop in West Asia and North Africa, while it
is sown in winter in South Asia. In Iraq, chickpea is traditionally grown in
Spring, but there are studies in the region for possibility of Winter sowing of
the crop particularly that contributed by the International Center for
Agricultural Research in the Dry Areas (ICARDA), Syria. Chickpea plays a
vital role in human diets in terms of protein (Table 1-1), has comparatively
high lysine content and it is also important in restoring soil fertility through
the symbiotic fixation of atmospheric nitrogen, particularly in dryland areas.
Chickpea is popular due to it’s multipurpose uses; culinary dishes of high
palatability, confectionery, and animal fed. Further, among the pulses,
chickpea has a unique property for it’s medicinal value against ailments. Its
acidic secretion, in the form of oxalic, malic, and acetic acids from the
glandular part of the leaf and pods, are used as medicine for colic and to repel
insects. Chickpea is also reported to reduce cholesterol accumulation in the
1
MSc. Thesis – Qasim Marzani
Chapter 1. Introduction
blood (Merchant,1984), while in the United states and Canada, Chickpeas are
almost entirely reconstituted and canned for use in salad bars(Muehlbauer and
Slinkard, 2000). The area cultivated under chickpeas in the world was 12.086
million hectare during the year 2000 with total world production of 9.393
million tones and the average yield was 777 kg/ha.
Table(1-1): Chemical components for chickpea seeds compared with the other
legume crops(Ali et al., 1990).
The structural evaluation for chemical components of seeds(%)
The crop
Water content
Ash
Protein
Oil
Fibers
Carbohydrate
Chickpea
9.7
2.7
19.1
5.1
6.8
56.4
Lentil
9.3
2.4
25.4
0.5
3.3
59.1
Mash
9.5
3.4
23.4
0.5
4.7
57.8
Cowpea
8.9
3.5
32.8
0.9
4.1
59.8
Bean
8.8
3.1
23.2
0.6
4.2
60.1
Majority of this production (8.437 million tones), was Asian product. In
Iraq, the cultivated area, production, and yield were 13000 ha, 8000 tone, and
640 kg/ha, respectively (FAO, 2000). While in Kurdistan region, the total
cultivated area during 2001-02 season was 180436.07ha distributed as
58661.70 in Duhok, 22690.60 ha in Erbil, and 99083.70ha in Sulaimani. The
total production was 96776 tones distributed as 34153 tone in Duhok, 11621
tone in Erbil, and 51002 tone in Sulaimani. The average yield in this region
was 536.4 kg/ha (FAO, 2002). Despite of it’s agronomic importance, the seed
productivity of chickpea is quite low (~700 kg/ha; Singh et al., 1994).
Chickpea yield production adversely affected by various biotic and abiotic
2
MSc. Thesis – Qasim Marzani
Chapter 1. Introduction
stresses, including cold, drought, insects, nematode, and fungal diseases
(Saxena and Singh, 1992). While, chickpea diseases play an important role for
reducing the yield, but the major disease which contributes to low production
in the world is the occurrence of the potentially destructive blight caused by
the fungus Ascochyta rabiei (Pass.)Lab., which was the most important foliar
disease of chickpea Reddy et al., 1992; Dolar, 1996). Sometimes Ascochyta
blight infections may cause 100% yield losses (Jimenez-Diaz et al., 1993). In
Australia, the disease has recently caused large crop losses which has resulted
in considerable reduction of land area grown to chickpea (Galvez et al.,2001).
Because of the importance of this disease in Kurdistan region, which caused
severe losses during the years 2000-02 in many chickpea growing areas, and
resulted in complete damages of the crop in some other locations, like some
villages in Ainkawa and Harir in Erbil province (personal observations and
communications) and because the disease has caused serious crop losses in
several countries and arrived sometimes 50 % annually and because there is
no suitable control measures recommended so far due to the diversity of the
pathogen, therefore this study was undertaken to:
1- Determine the geographical distribution of the disease in Erbil
province.
2- Find out the sources of inoculum.
3- Study of physiologic races or pathotypes of the pathogen.
4- Indicate the favorable conditions for epiphytotics.
5- Assert the effect of the disease on yield quantity and quality.
6- Study the control measures, including agricultural and chemical
control, resistant cultivars and integrated method.
3
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
Chapter 2 LETIRATURE REVIEW
2.1 Chickpea diseases
More than fifty diseases are known to infect chickpeas (Nene, 1980).
According to Grewal (1988), Ascochyta rabiei, Botrytis cinerea, Fusarium
oxysporum f.sp.cicers, Macrophomina phaseolina, Sclerotinia sclerotiorum,
Operculela padwickii, and Uromyces ceceris-arietina, are the main pathogens
infect Cicer arietinum, while Haware (1990) reported that Ascochyta blight
(A. rabiei), Fusarium wilt (Fusarium oxysporum f.sp.cicers), and stunt (bean
leaf roll luteovirus) in the Mediterranean area, as well as stem rot (Sclerotinia
sclerotiorum) and root rot (Macorophomina phseolina) are commonly
observed in the farmers fields. In Iraq, there were very few studies on
chickpea diseases, soft root rot (Fusarium solani) and dry root
rot(Rhizoctonia bataticola) by Al-Talib (1988), Ascochyta blight(A. rabiei)
by Al-Taee(1997), and Fusarium wilt (Fusarium oxysporum) by Al-Taee
(1999) were reported in Mosul Governorate only. While in Kurdistan region,
despite the importance of the Ascochyta blight, there were no studies on this
devastating disease. Ascochyta blight caused by A. rabiei was a serious
disease on chickpea fields which might cause 100 % yield losses in severely
affected fields especially, with the susceptible cultivars (Nene, 1984).
2.2 Geographical
distribution
of
Ascochyta
blight
disease
The disease, Ascochyta blight, was first observed in the North-west
Frontier province of British India, now in Pakistan(Butler,1911). It was
reported from 31 countries(Nene et al., 1989), it was observed in
Morocco(Atnasoff and Kovachevski, 1929), France(Labrousse, 1930),
Greece(Sarejanni, 1939) , Palestine(Halfon-Meiri, 1970), Iran(Kaiser, 1972),
4
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
Canada(Morral and Mackenzie,1974), and in Australia(Cother, 1977), but in
Iraq, the first report was by Al-Baldawi et al.(1979), while in Egypt by AbdelMonem(1983). In the United States, the disease first was reported also in
1983(Kaiser and Muehlbauer, 1984), and in California in 1994(Guzman et al.,
1995).
The disease has recently been found in commercial chickpea
crops in Victoria, Australia(Nasir and Bretag, 1997), and in Latin America,
Bolivia, in 1999(Kaiser et al., 2000).
2.3 The economic importance of the disease
The disease has caused serious crop losses in several countries in the
past and is continuous to take its toll. In Pakistan, where chickpea was the
major pulse crop, experienced with many epiphytotics, Sattar(1933) reported
an annual loss of 25-50% of the crop since 1922, also during 1978-82 the
disease was reduced yields by one half(Saleem,1984). India is also
experienced with this destructive disease. Though, according to Reddy
(1984a), in northern parts of India losses of about one million tones were
suspected due to the outbreak of blight during 1981/1982 season. Gaur and
Singh(1993b) reported that the Sri Ganganagar district of Rajasthan, India,
experienced three severe epiphytotics of chickpea Ascochyta blight(A. rabiei)
during 1980-81 to 1982-83 and irrigated crops closer to western part of the
district adjoining Pakistan suffered losses of 77-84% while minimum loss
were 20-30% in the South-east corner of the district bordering Haryana state
and Bikaner and Churu districts of Rajasthan(were chickpea is grown under
rainfed cultivation). The overall estimated loss in the district under two
epiphytotics(1981-82 and 1982-83) was 51% reducing yield to 321kg/ha
compared with yields of 655kg/ha in disease free years. According to
Kovachevski(1936a), 20-50% of the crop was lost annually in Bulgaria, with
occasionally total losses in some fields. Sometimes Ascochyta blight
5
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
infections may cause 100% yield losses (Nene and Reddy, 1987; JimenezDiaz et al., 1993; Acikgoz et al., 1994 ). According to Demetriads et
al.,(1959), the crop damage in Greece was 10-20% during 1957-58 and in
Spain, according to Puerto-Romero(1964), the disease caused great losses of
chickpea wherever it was grown in Spain, and in some years inspite of Spring
sowing the losses reached to 100%, on the other hand , in Italy, inspite of
Winter sowing, the losses reached to 100%, while in the United states, there
were several reports about the losses, in northern Idaho, according to Wiese et
al.,(1995), more than half of the crop was destroyed by Ascochyta blight in
1987, also in the same year, Kaiser et al.,(1994) reported the yield loss due to
primarily to Ascochyta blight and resulted in financial losses of over 1 million
US$ , while Guzman et al.,(1995) recorded 30% of 1.5 acre field was heavily
infected after cool and rainy weather during May 1994 on experimental lines
growing in Frenso country, California, United states.
In Australia, the disease has recently caused large crop losses which
has resulted in considerable reduction of land area grown to chickpea (Galvez
et al.,2001). This disease also causes severe losses in the Mediterranean basin,
damage of 30% was estimated during 1982 in northern Syria(Reddy,1984a).
Labrousse(1930) had reported Ascochyta blight to be very destructive in
Morocco in 1929, also Reddy(1984a) stated that the chickpea production was
reduced during 1976-78 in Morocco. The disease was first recorded in Iraq
and 30% infection was estimated also (AL-Beldawi et al.,1979).
2.4 Disease symptoms and development
The disease symptoms appear on all foliar parts(Wiese et al. 1995;
Nasir et al., 2000; Hamid &Strange 2000). On leaves and pods, the spots are
circular with the dark pycnidia arranged in concentric rings, while on the
stems, the lesions are elongated and often cause girdling. The entire foliage
6
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
can be blighted resulting in death of the plants(Reddy, 1984e). When the
whole stem girdling occurs, the portion of the plant above the point of attack
rapidly dies and in the early stages of the disease tender branches topple over.
If the main stem is girdling near the collar region, the whole plant dies. On
green pods, the lesions are usually circular with dark margins and the
pycnidia are arranged in concentric circles and in severe cases, the fungus can
infect the seeds, which become shriveled, and dark. When pods infected in the
early stages of the developments, they become blighted and produce no seeds
or black shriveled seeds(Saleem, 1984;Reddy, 1984a). On the other hand,
Guzman et al.,(1995) observed symptoms on the plants in the field included
tan to brownish lesions on leaves, stems and pods, with brown to black
pycnidia immersed in the host tissue and arranged in concentric rings within
the lesions. Only heavily infected seeds will bear visible blight symptoms,
which include small size wrinkles, lesions, and/or dark discoloration(Wiese et
al., 1995). According to Porta-Puglia et al.,(1996), the symptoms on the
leaves appeared as a circular spots, soon followed by drying of apart or the
whole lamina. On the stems, more or less extensive lesions were observed,
ranging from flecks to larger lesions(>5mm2), which in the case of severe
attacks evolved in to complete and deep girdling. Hamid and Strange(2000),
who had reported that the disease caused epinasty of petioles and young
branches followed by water-soaking and necrosis. When the stems and
petioles are girdled, they usually break.
According to Reddy(1984a), in the field, the disease usually appears
around pre-flowering to flowering in small circular patches which rapidly
increase under favorable weather conditions. The seedlings from infected
seeds show dark brown lesions near the collar region and sometimes show
damping-off symptoms. Initially, small, round, white necrotic specks appear
on newly formed leaves of susceptible cultivars, and under favorable
7
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
conditions the lesions expand rapidly and coalesce cause the blighting of the
buds. Lesions on stems and petioles are brown, elongated(3-4cm), bear
pycnidia in form of black dots. Chauhan and Sinha(1971) showed that the
lesions in young plants were larger and the symptoms appeared sooner and
under continuous light lesions were smaller and sporulation was markedly
reduced.
According to Pandey et al.,(1987) the pathogen penetrated directly
through the epidermis, through stomata. Penetration occurred in three days,
no symptoms being apparent until day four when yellow specks on the leaves
indicated the presence of sub-epidermal mycelial aggregates. At 4-5 day these
enlarge and start to differentiate, and forming pycnidia. Mature pycnidia were
seen as black dots on the surface of the leaves after 5-6 days. The sizes of the
yellow lesions were increased and host tissues showed necrosis six days after
inoculation, coinciding with pycnidial maturity. While Gurdip et al.,(1988)
showed that the disease symptoms appeared seven days after inoculation. The
time course of infection, development of leaflets and stems of susceptible(ILC
1929) and resistant(ILC 3279) plants were monitored by Hohl et al.,(1990)
using light microscopy or Scanning Electron Microscope(SEM), so according
to this report, spores of A. rabiei began to germinate from 12h post
inoculation(hpi) and developed a polar germ tube; fungal colonization,
secretion of a mucilaginous exudate and appressoria formation 1-3 day post
inoculation(dpi) were identical on both cultivars, and leaves of susceptible
plants were invaded by the fungus directly through the cuticle, the fungus
then spread subepidermally followed by a rapid collapse of the leaf tissue(4-6
dpi), then development of leaf spots and fungal pycnidia could be observed 68 dpi. Trapero-Casas and Kaiser(1992b) showed that at constant temperature
of 20oC the minimum incubation and latent periods were 4.5 and 5.5 days,
respectively. Lower or higher temperatures increased the duration of these
8
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
periods, while Iqbal et al.,(1994) observed that the symptoms developed 5-6
days and susceptible genotypes died within 10 days of inoculation.
2.5 The causal organism
2.5.1 Identification of the pathogen
A. rabiei (Pass.) Labr. is the causal agent of Ascochyta blight of
chickpea and it is considered as one of the most important disease on
chickpea limiting the production of the crop(Reddy et al., 1992; Singh and
Reddy, 1993; Porta-Puglia et al., 1996; Nasir et al., 2000; Galvez et al.,
2001).
The first identification of the fungus was under the name Zythia rabiei
Pass. by Passerini in 1867 depending on non-septated pycnidiospores, but
after that, Comes in 1891 gave the name Ascochyta pisi to the causal
fungus(Khune and Kapoor, 1980). Then, after sufficient study of Saccardo
system for classification of fungi by Trotter, the fungus classified as
Phyllostica rabiei(Pass.)Trott. and both Labrousse(1930) and Spargue(1930)
concurred with Trotter’s classification due to non observing of the bi-celled
pycnidiospores, but in 1931, Labrousse did not agree with Trotter about the
name of the fungi, so he gave the name A. rabiei(Pass.) Lab. to the pathogen
depending on his observation of the presence of the bi-celled pycnidiospores
which ranged 2-4%, and the latter name was the most common and
compatible with the nomenclature of British Institute of fungi (Nene et al.,
1989). Nevertheless, Reddy(1984a) argued that the taxonomy of the fungus
causing blight is uncertain and it has been called A. rabiei , Phyllostica rabiei
and Phoma rabiei, but A. rabiei is the most common and widely accepted
name for the fungus containing some bi-celled spores(2-4%), while Phoma
rabiei has single celled spores. Since Phoma spp. can also have bi-celled
spores(up to 5%), it suggested to be called Phoma rabiei. The author(Reddy)
9
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
claimed that he has seen, in some samples of blight, bi-celled spores to the
extent of 20%. Despite of the above-mentioned reports, Guzman et al.,(1995)
had shown that the conidia of the blight pathogen were usually non-septate,
straight and hyaline.
According to Alexopoulos et al.,(1996), the fungus Ascochyta rabiei
(anamorph), classified within ascomycete fungi as
Mycosphaerella
(Teleomorph) as follow:
Kingdom: Fungi
Phylum:
Ascomycota
Class:
Filamentous ascomycetes
Series:
Loculo ascomycetes (ascomycetes with ascostroma)
Order:
Dothideales
Genus:
Mycosphaerella
Species:
rabiei
2.5.2 The sexual stage
The first observation of the sexual stage of A. rabiei was in Bulgaria in
1936 by Kovachevski(Kovachevski, 1936b). Later it was confirmed in
Russia(Gorlenko and Bushkova, 1958), Greece(Zachos et al.,1963), Hungary
(Kovics et al.,1986), Syria(Haware, 1987), Spain (Jimenez-Diaz et al.,1987),
United states(Kaiser and Hanan, 1987), Turkey(Kaiser and Kusmenoglu,
1997), and in Iraq(Al-Taee ,1997).
Although Didymella rabiei, the perfect stage of A. rabiei, has been
reported from many regions of the world(Nene and Redy, 1987), but it ’s
influence on disease epiphytology and pathogen diversity is still mysterious.
The Teleomorph might be more widespread than previously thought and the
airborne ascospores may serve as primary inoculum(Kaiser and Muehlbaur,
10
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
1988; Kaiser et al., 1994; Nasir et al., 2000). The Teleomorph, Didymella
rabiei(Kov.)v.Arx, is a bipolar heterothallic ascomycete (Dothideales). The
two mating types which are required for sexual reproduction are designated as
MAT-1 and MAT-2(Wilson and Kaiser, 1995; Kaiser and Okhovat, 1996;
Kaiser et al., 1998). However, the presence of both mating types in a
population does not necessarily mean that this population is sexually
reproducing, because certain conditions in the microenvironment may prevent
mating of compatible isolates(Geistlinger et al., 1997).
Pseudothecia(the sexual fruit body) on chickpea residue are visually
similar to pycnidia, but ascopores are easily distinguished from conidia
(Punithalingam and Holliday, 1972). Ascospore size was 12-22µm× 4.7-6µm,
and ascus dimensions were 48-85µm×8-22µm(Kovachevski;1936b; TraperoCasas and Kaiser, 1992a;).
The Teleomorph may contribute of long distance spread of the disease
by airborne ascospores of the pathogen(Armstrong et al., 2001). The
discovery of the Teleomorph helps to explain how the pathogen is spreading
in the area, where chickpeas have never been grown before and where
apparently clean seeds had been planted(Kaiser and Hannan, 1987). It is
suggested that the Teleomorph may play an important role in increasing
genetic diversity in the pathogen(Akem, 1999; Armstrong et al., 2001).
Nevertheless, in some countries, the Teleomorph has not been yet found. In
Australia, according to Khan et al.,(1999a), the Teleomorph has not been
found and the results to date suggest that only one mating type is present. It is
suggested that quarantine restriction on imported chickpea seed should be
retained to prevent the introduction of opposite mating type.
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2.5.3 Physiologic races, pathotypes and isolates of A.
rabei
The investigations for presence of races started when the varieties which were
released earlier as resistant to the disease became susceptible(Qureshi, 1984b;
Singh and Reddy, 1991; Jamil et al., 1993). Therefore, several researchers
investigated the existence of races in A. rabiei. Lurtha et al.,(1939) and Arif
and Jabbar(1965) observed much variability in the size, growth, sporulation,
and other cultural characteristics of fungal isolates, but they did not find any
evidence of races existence . Eleven races were first reported from the state of
Punjab in India(Bedi and Aujla, 1969). Subsequently, two races were reported
in India and six in Syria and Lebanon(Vir and Grewal, 1974a, Reddy and
Kabbabeh, 1985; Porta-Puglia, 1990). Another approach of races were
reported in India by Gurdip and Singh(1990) when they identified twelve
races by using 12 differential chickpea cultivars utilizing 348 isolates
collected from different parts in India. New races have also been identified in
Italy by Porta-Puglia et al.,(1986), and also, three groups of isolates have been
identified in Italy by Porta-Puglia et al(1996) when he used 41 Italian isolates
which differentiated by using 13 chickpea genotypes. In Palouse region of
northern Idaho, USA, eleven different virulence forms were distinguished
among 39 isolates by using 15 differential hosts(Jan and Wiese, 1991), and in
Turkey, the first report of existence of races was in 1992 when three races
were found by Dolar and Gurcan(1992) by using 6 differential chickpea
cultivars. In Pakistan Jamil et al.,(1995) had reported presence of eight
pathogenic groups among 102 isolates, collected from different parts of the
country during 1984-92 by using 11 chickpea differentials. In Iraq, four races
were distinguished by Al-Taee(1997) among 31 isolates collected from
different locations of Ninavah province by using 12 differential cultivars, and
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in Iran, among 400 isolates collected from different provinces, two
physiologic races were distinguished (Noorollahi, 2000).
According to Reddy et al.,(1983), resistant genotypes in Syria became
susceptible when they were grown in Pakistan, this indicates the presence of
physiological races of the fungus. In addition to the presence of races, isolates
of A. rabiei can be grouped into pathotypes based on to the virulence
aggressiveness, by using plant material accessions ILC 3279(resistant), ILC
482(tolerant), and ILC 1929 (susceptible) as host differential set for testing
the virulence of A. rabiei isolates. This internationally used standard was
obtained from ICARDA, Aleppo, Syria(Jamil et al., 2000). In pathogenicity
assay for isolates of A. rabiei from different chickpea growing areas of Syria,
the analysis revealed the occurrence of three distinct pathotypes. The
pathotype I [least aggressive], the pathotype II [medium aggressive], and
pathotype III [highly aggressive] (Udupa and Weigand, 1997; Udupa et al.,
1998). The same results were obtained by Jamil et al.,(2000) within 130
isolates from Pakistan. Moreover, In cluster analysis of virulence, Hamza et
al.,(2000) found five highly virulent pathotypes and they showed that
virulence instability in Tunisia, was probably due to the utilization of non
resistant cultivars and the occurrence of sexual reproduction between the
isolates.
According to Gowen et al.,(1989), the pathogenicity of some isolates,
particularly, those from the Indian Subcontinent and Western Asia, was
greater than that of most isolates collected in the western Mediterranean.
Porta-Puglia(1992) who found that pathogenic groups of A. rabiei were
determined by recording the severity of infection symptoms of different
chickpea cultivars. Mmbaga et al.,(1997) investigated the pathogenic
variability of A. rabiei in a single race is a factor of both race and
aggressiveness and that the two coexist.
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2.6 Epiphytology of the disease
The Ascochyta blight of chickpea is typically epiphytotic and many
epiphytotic forms were recorded worldwide. In Pakistan, that chickpea is the
major pulse crop, has experienced several epiphytotics starting in 1915
(Qureshi, 1984a). Kauser(1965) traced the history of gram blight epiphytotic
in Pakistan from 1928 to 1959. In the blight epiphytotic of 1956/57, he
observed blight infection on early sown crop in late November in Attok, and
December in Rawalpindi, the blight broke out again in 1957/58 crop season,
blight developed severely during January and February. Another epiphytotic
forms started in the 1978/79 cropping season and for the fourth consecutive
year(1981/1982) the disease has devastated the crop(Qureshi, 1984a).
India also experienced with blight disease and three severe epiphytotics
of chickpea Ascochyta blight(A. rabiei ) were recorded during 1980/81 to
1982/83(Gaur and Singh, 1993b). Guzman et al.,(1995) observed a severe
blight of chickpea during May, 1994 in Frenso Country, California, USA,
after cool and rainy weather.
When suitable conditions persist for long periods, the disease becomes
epiphytotic and causes severe damage to the crop especially to susceptible
cultivars, and the disease favored the cool and humid weather[15-25oC and
>150mm rainfall](Sattar and Hafiz, 1952; Kauser, 1965; Reddy and Singh,
1990b; Akem, 1999). The disease initially appears in small areas within
affected fields and spreads rapidly when the cool and wet conditions
prevail(Kaiser, 1973; 1995). The wind which accompanied by rain carrying
spore splashes and broken diseased tissues, spread the disease; thus accelerate
dispersal as well as development of disease(Dey et al., 1994a; Santra et al.,
2001).
According to Reddy and Singh(1990b), the epiphytotics was developed
when weekly mean temperature were between 10-20oC, while weekly
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maximum temperature more than 25oC slowed disease progress and weekly
mean relative humidity >60% promoted blight. On the other hand, Ketelaer et
al.,(1988) had reported that the monthly average temperature of at least 8 oC
and monthly precipitation of at least 40mm is necessary of an epiphytotic of
this disease of Cicer arietinum , on the other hand Abdou et al., (1991b)
showed that the optimum temperature for growth of A. rabiei was 20oC.
According to Jhorar et al.,(1998a, 1998b), disease severity increased with
wetness duration according to an exponential asymptote, with maximum
value after approximately 8h, and infection was significant when the dry
leaves were exposed to 98% relative humidity for 48h, further he confirmed
that the average of relative humidity and maximum temperature during the
crop season were good indicators of the epiphytotic conditions in the crop
field, while Jhorar et al.,(1997) observed that the humid thermal ratio(HTR),
was best fitted by a quadratic function, and they stated that predications of the
disease over a twelve years period showed that there was better
agreement(less scatter) between the predicted and observed values with HTR
than either maximum temperature or relative humidity alone. Therefore the
use of HTR as a basis for predictive scheme to advise on fungicide
application is suggested. On the other hand, Gaur and Singh(1993b) revealed
that the inoculum direction from the western side influenced the epiphytotics,
also Pederson and Morall(1995) found horizontal disease spread was more
extensive from West to East than the opposite direction, while Guar and
Singh(1993a) had noted that crops sown in North-east to South-west rows had
the most blight, as wind direction was usually from the North-west.
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2.7 Sources of infection
2.7.1 Infected seeds
One of the major sources of primary inoculum is the infected chickpea
seed(Ilyas, 1984; Abdel-monem, et al., 1984; Maden, 1987; Singh and Reddy,
1990; Kaiser et al., 1994; Bhutta, 2000), and it is important in the long
distance spread and survival of the Ascochyta blight and usually introduces
pathogen inoculum to new areas through many countries worldwide by seed
transmission among these areas or countries(Halfon-Meiri, 1970; Kaiser,
1997).
In the infected seeds the pathogen located primarily on/or in the seed
coat and in very few cases penetrated into the cotyledons and, rarely, to
embryo(Dey et al., 1994b; Al-Taee, 1997 ), while Shakir and Mirza(1994)
showed that A. rabiei isolated only from cotyledons, and infected seeds
usually produce seedling infection.
In pot tests, Vishunavat and Chaube(1986) showed that seedling
infection was 2% when were stored at room temperature, compared with 48%
for seeds from cold storage, on the other hand, Dey et al.,(1994b) had noted
that seed infection resulted from both externally and internally seed-borne
inoculum and the transmission from naturally infected seeds to seedling was
25% in greenhouse and 12.2% in the field.
According to Maden et al.,(1975), 70% of seed infection was recorded
in Turkey, while Grewal(1982) had noted that seed infection ranged from 3 to
17.5% for the desi varieties and from 7.5 to 60.5% for the kabuli ones, and
according to Kaiser(1972), A. rabiei was isolated from seeds which had been
stored for more than 117 weeks at Safiabad, Iran, where Summer
temperatures exceeded 45oC, and at ICARDA, the fungus survived in 65% of
infected seeds stored for two years at room temperature(Reddy, 1984d).
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Vishunavat and Chaube(1986) who found that seed infection declined after
storage at room temperature from 70% in May to 3% in December, while
Tripathi et al.,(1987b) observed that the fungus survived 14-15 months in
infected Cicer arietinum seeds stored at 5-10oC and for 12 and 10 months at
20 and 30oC, respectively, and viability of A. rabiei declined, in seeds kept
at room temperature, at constant rate and was not hastened by very high
Summer temperature(37-40oC). Gaur and Singh(1996) revealed that viability
of A. rabiei spores decreased with the increase of storage periods, from 74%
in May to 17% in November, during 7 months of storage at room temperature
(3.6-38.8oC).
2.7.2 Infected chickpea debris
The plant debris that lay in the field, is an important source of inoculum
which may initiate new centers of infection from rain splashed conidia
occurring an epiphytotic form(Ilyas, 1984; Abdel-Monem, et al., 1984, Kaiser
et al., 1994; Dey et al., 1994a). According to Lukashevich (1958b), after the
death of chickpea plants, the pathogen will live as a saprophytic fungus on
plant debris and when the next season comes, the fungus form many
quantities of inoculum.
Kaiser(1973) found that the fungus survived over 2 year in naturally
infected tissue at 10-35oC, RH 0-3% on the soil surface, but it loss ’s it’s
viability rapidly at RH 65-100% at soil depth 10-40 cm, while Nene and
Reddy(1987) revealed that the fungus can be viable on plant debris for only 8
months and losses it’s viability within 4 months when the plant residues
buried at depth 10 cm, on the other hand, in USA, Kaiser et al.,(1987)
observed that the fungus remain viable on the infected pods and stems for 57
and 81 week, respectively, and it loss’s it’s viability after 10 and 15 week,
respectively, when the residues buried at depth of 16cm, also Singh et
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Chapter 2. Leterature Review
al.,(1992a) found that the fungus remain viable on plant debris for four
months and lose it’s viability when buried at depth of 10cm. Al-Taee(1997)
reported that no infection was occurred when plant residues buried at depth of
5cm or more, and according to Wiese et al.,(1995) the fungus survives in
infested crop residues as long as they remain visible on the soil surface.
In addition to the pycnidia producing (asexual stage), the plant debris
may produce the pseudothecia (sexual stage), the source of airborne
ascospores that may act as primary inoculum for infection. Trapero-Casas, et
al.,(1996) reported that the airborne ascospores of Didymella rabiei is major
primary inoculum of Ascochyta blight epiphytotics in chickpea crop in
southern Spain, which formed on infected chickpea debris laying on the soil
surface during Autumn-Winter sowings.
2.7.3 Host range (alternative hosts)
The previous thought which considers chickpea(Cicer arietinum L.) the
only host of the pathogen A. rabiei, is not acceptable now everywhere.
Although, some of researchers revealed that A. rabiei was host specific, like
Reddy(1984d) who investigated that only chickpeas are susceptible to A.
rabiei. Abdou et al.,(1991b) also studied legumes host ranges of the pathogen
and found that only chickpea was host of the pathogen. Similar results were
obtained by Tripathi et al.,(1987a) when they reported that none of 44 crop
and weed species occurring in Tarai of Nainital, India, became infected with
the pathogen in greenhouse inoculation tests and they concluded that the
Pantnagar isolates of A. rabiei is host specific. Furthermore, Kaiser et
al.,(1998) observed that both cultivated and wild chickpeas(Cicer montbretii)
are infected naturally by A. rabiei in Bulgaria. Nevertheless, some other
researchers obtained different results, like Kaiser(1973) who investigated that
the fungus also infected phaseolus vulgaris and cowpea on inoculation with
18
MSc. Thesis – Qasim Marzani
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isolates from India and Iran, but was highly pathogenic only to Cicer
arietinum. Nene and Reddy(1987) also observed the infection of phaseolus
and pea in greenhouse inoculation, but Kaiser(1991) who observed reddish,
brown lesions, on inoculation with spore suspension, on several plant species
and pycnidia developed in necrotic tissue of Lucerne(alfalfa) and sweet white
clover. The Teleomorph(Mycosphaerella rabiei) developed also on over
wintered tissue of sweet white clover and pea that had been inoculated with
conidial suspension of compatible isolates, meanwhile, single-ascospore
isolates were highly pathogenic to chickpea. Kaiser(1992) who could isolate
the pathogen from alfalfa, phaseolus, pea, and cowpea in USA, and argued
that these hosts considered secondary ones of the fungus and may play an
important role in life cycle of the fungus.
According to Montorsi et al.,(1992), A. rabiei was isolated from
barseem clover(Trifolium alexandrium ) seeds from Foggia, Italy. Barseem
clover and chickpea seedlings were inoculated with conidial suspension of the
isolated fungi and disease symptoms appeared after 10-12days, further, the
fungus was re-isolated from both hosts. They concluded that hosts other than
chickpea have a role in the epiphytology of A. rabiei, while Gaur and
Singh(1993a) found that inspite of the ability of the local isolate to infect
leaves of cowpea, French bean(Phaseolus vulgaris) and pea(Pisum sativus
L.), no pycnidia developed on the lesions and no infection were produced on
stems or petioles. Khan et al.,(1999b) who inoculated 20 plant species with an
isolate of A. rabiei obtained from chickpea in south Australia, found that the
isolate was pathogenic to chickpea and 4 common bean(P. vulgaris)
cultivars(Brown boy, CH-190-70D, Cran-34, and rain-bird). None of the other
plant species was infected by the pathogen.
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2.8 Control methods
2.8.1 Agricultural methods
2.8.1.1 Control by changing date of sowing
Chickpea is a spring sown crop in west Asia and north Africa, while it is
sown in winter in South Asia, and in Mediterranean region, chickpeas are
traditionally sown at the beginning of Spring(March-April), therefore farmers
save their crop from Ascochyta blight damage, but seed yield is greatly
reduced because of the moisture and heat stresses(Haddad, 1981; Keatinge
&Cooper, 1983; Singh &Reddy, 1990), and by Spring sowing they also save
their crops from Winter weeds(Solh and Pala, 1990). According to
Singh(1990), the lack of resistant cultivars to Ascochyta blight and cold
tolerant cultivars both stresses appear to be the main reason for not cultivating
chickpea during Winter in Mediterranean region.
Hawtin and Singh(1984) noted that the yield of chickpea can be
increased by sowing in Winter(early December) provided that Ascochyta
blight is controlled. According to Rheenen(1991), in West Asia and
Mediterranean region, drought avoidance by Winter sowing has been
achieved by incorporating disease resistant and changing the sowing date ,
this has resulted in a 75% yield increase, while Singh et al.,(1997) showed
that sowing in Winter with cultivars tolerant to cold and Ascochyta blight
produced 70% more seed yield than the Spring sown crop, also, Winter sown
plants were taller than those sown in Spring, permitting harvesting by
combines. Iliads(1998) found that yields were consistently higher, between
26-330%, with Autumn sown crops than the Spring sown crops and higher
yields in Autumn were produced only in resistant to A. rabiei and Winter
frosts, but Toker and Cagiran(1996) had reported that an epiphytotic form of
Ascochyta blight was occurred when sowing date changed from Spring to
20
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Chapter 2. Leterature Review
Winter, although yield of chickpea is increased. Tufail(1984) who stated that
the date of planting and stand establishment are the most important factors
that determine chickpea yield especially in rainfed areas. In Syria and
according to Pala and Mazid(1992), advancing the sowing date from Spring
or late Winter to early Winter resulted in substantial yield increase, also in
Morocco, the potential yield is about 2 tone/ha in Winter chickpea and 0.6
tone/ha in Spring chickpea when this Winter chickpea is 25-45 day earlier
than Spring chickpea(Kamel, 1990).
According to the report by Abdou et al.,(1991a), the incidence of A.
rabiei infection was high on chickpea plants which were sown early(Oct.6th),
and sowing dates from Oct. 20 to Nov. 30 reduced A. rabiei infection. In trials
with 4 sowing dates, Eser et al.,(1991) who observed yields were highest with
the earliest date(end of Feb.) under Ankara conditions in Turkey, but early
sown plants were more susceptible to A. rabiei. Muehlbauer and
Slinkard(2000) had found that Winter chickpea has been a factor for increased
production in California, USA. While in Cyprus, Spring sown have given
very low yields(Hadjichristodoulu &Gaborcik, 1996).
In a field trial conducted in Iraq during 1985/86 season by cultivating
ILC 482, ILC 3279, and some other cultivars in Sulaimni, this trial proved
that Winter sowing was possible throughout the Mediterranean region(Singh,
1990), he also added that a minimum of 500kg/ha additional yield can be
obtained by changing the sowing season from Spring to Winter.
2.8.1.2 Control by production disease-free seeds
Since infected seed is the major source of primary inoculum, so the
production and the supply of the disease-free seeds is essential for the control
of blight. Production of seeds in drier zones where blight does not appear is
ideal(Reddy, 1984a). Production of healthy seeds also can be achieved under
favorable conditions for blight development, by using a foliar spraying
21
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Chapter 2. Leterature Review
(Saleem, 1984). On the other hand, Kaiser(1984) suggested that chickpea
seeds can be produced with cultivating in arid environments, crop rotation,
and by field sanitation and these practices can significantly reduce or
eradicate the disease in infested areas. Wiese et al.,(1995) stated that seeds
look healthy may in fact be infected with low levels of A. rabiei, thus the
growers should be certain that their seed come from fields and areas that are
free of Ascochyta blight. Using certified or foundation seed should also
provide some assurance that blight was either not present or not detected in
the present seed fields.
2.8.1.3 Control by agricultural practices
There were several agricultural practices that prevent build-up of A. rabiei, so
that chickpea should be grown in rotation with other crops, such as cereals to
prevent build-up of A. rabiei on any infested debris, and certain practices such
as plowing, speed up debris decomposition (Wiese et al., 1995). Gaur and
Singh (1995) observed that summer plowing in fall season found to reduce
infection comparing with keeping the land flow. Field sanitation by burning
the disease debris or burying the debris at 10 cm or more, were also suggested
to prevent new centers of infection to appear (Reddy, 1984a; Gaur & Singh,
1995). On the other hand Wiese et al.,(1995) who suggested that chickpea
crops should not be grown on the same field site more frequently than every 3
or 4 years, furthermore, successive chickpea crops should not be grown near
fields that were infested with blight during the previous year. Gaur and
Singh(1993a) found a progressive decrease in the above-ground infection and
increase in under-ground infection by A. rabiei occurred with an increase in
depth of sowing, and no symptoms appeared on the above-ground plant parts
when the seed was sown at depth of 15 cm or more. Also, in greenhouse
experiment, Al-Taee(1997) stated that burying the infected plant debris to
5cm or more will prevent plants from infection.
22
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Chapter 2. Leterature Review
2.8.2 Control by planting resistant cultivars
The cultivation of the resistant/tolerant cultivars appears to be the best
method and the most economically and environmentally sound means to
control Ascochyta blight of chickpea(Malik, 1984a; Porta-Puglia et al., 1996;
Acikgoz, 1997; Carvalho et al., 1999). The first report of resistant to
Ascochyta blight was in 1931(Anonymous, 1931) and it was found in wild
chickpea [Cicer reticulatum ] (Anonymous, 1977), while the first resistant
cultivar released for cultivation was F8 in Pakistan in(Lurtha et al., 1938).
Progress in breeding blight-resistant cultivars has been hampered by the
absence of dependable sources of resistance(Singh & Reddy, 1993).
Nevertheless, there is some research centers in the world for producing
chickpea resistant cultivars. Relying on the results obtained from the On-Farm
trial, Syria, in cooperation with ICARDA, has released two cultivars, Ghab-1
(ILC 482) the high yield, wide adaptation, and moderately resistant, and
Ghab-2(ILC 3279) the tall type, cold tolerant and resistant to Ascochyta
blight(Singh, 1990). The Nuclear Institute for Agriculture and Biology
(NIAB), Faisalabad, Pakistan, was also initiated a programme aimed in the
induction of new sources of resistance and improvement of resistance against
Ascochyta blight of chickpea caused by A. rabiei. The significant
achievements of this programme were the evolution of blight resistant, high
yielding varieties(CM 72 in 1983, and CM 88 in 1994), thereby the release of
these disease resistant varieties has helped to stabilize chickpea production in
Pakistan(Haq et al., 1997), and also efforts by USDA Agricultural Research
Service Scientists at Pullman, Washington, led to recent release of the blight
resistant varieties Sanford and Dwelly(kabuli- type) in 1993 and Myles( desitype) in 1994, and their use is recommended especially in blight prone or high
moisture areas(Wiese et al., 1995). According to Haq et al.,(1999), chickpea
variety CM98 which was derived from variety K850 by induced mutation
23
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
using gamma-radiation, have seed yields of 1559Kg/ha, an increase over 20%
compared with control varieties, further, it is resistant to A. rabiei
and
Fusarium oxysporum.
Due to the evolution of new physiologic races of the Ascochyta blight
pathogen, a resistant cultivar will eventually become susceptible, this
happened in Pakistan when the chickpea cultivar F8 released in 1938, became
susceptible and was replaced by C12/34 in 1946(Singh, 1984), and because of
numerous races in A. rabiei, would be difficult to cultivars that are resistant
across all locations(Singh & Reddy, 1990). Thus, in cultivars were vertical
resistant does not last long, due to rapid changes in pathogens, the horizontal
resistance is favored(Malik, 1984a). The absence of durable blight in the field
has been attributed to the appearance of new pathogens(Singh et al., 1992a)
and high levels of polymorphism in aggressiveness in pathogen populations
(Vir & Grewal, 1974a; Reddy & Kabbabeh, 1985; Malik & Rahman, 1992;
Porta-Puglia, 1992).
Many researchers argued the inheritance of Ascochyta blight resistance.
Hafiz and Ashraf(1953) were the first who report that the inheritance of
Ascochyta blight-resistance was dominant and monogenic. Acikgoz and
Demir(1984) they also showed that one gene was responsible for resistance,
but they said that this gene could be recessive or dominant. Some other
reports indicate that resistance to Ascochyta blight in chickpea is conferred by
more than one gene(Singh and Reddy, 1983; Tewari & Pandey, 1986;
Muehlbauer & Singh, 1987; Singh & Reddy, 1989; Kusmenoglu, 1990;
Muehlbauer & Kaiser, 1994), and recently, Takeoglu et al.,(2000) showed
that two complementary recessive genes with several modifiers conferred
Ascochyta blight resistance. The absence of one or two of the major genes
confers susceptibility, whereas the presence of the modifiers determines the
24
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
degree of resistance. They concluded that the locations of the genes
conferring resistance are not known.
Hari et al.,(1988) found that leaves of cultivar E100Y(M) and pods of
E100Y and stem segments of both resistant cultivars had more hairs than the
susceptible varieties and the number of non-glandular hairs on the leaves was
much higher than glandular hairs, whereas the reverse was true for stems and
pods. On the other hand and according to Vogelsang and Barz(1990) after
treatment with an elicitor derived from the pathogen A. rabiei, the cells of the
resistant line ILC 3279 contained 5-fold higher level of chitinase activity in
comparison with susceptible line ILC 1929 cell culture. They concluded that
the accumulation of extracellular hydrolase activity might play an important
role among the various plant defense mechanisms previously determined for
the incompatible interaction between the resistant cultivar and it’s fungal
pathogen.
The inhibition of A. rabiei development was also studied by Dolar and
Gurcan(1993) when they found, after inoculation with A. rabiei, that the
contents of medicarpin and maachiain(phytoalexins) were sufficient for
inhibition of fungal development in resistant cultivar, but insufficient in the
susceptible one and the presence of maachiain only in resistant cultivar
indicates it’s important role in the resistant. Phenolic compounds were
another inhibiting agent which formed after inoculation with A. rabiei in
resistant genotype cells more than the susceptible ones(Khirbat & Jalali,
1997; Anil et al., 1998). Khirbat and Jalali(1998) also observed the post
infection anti-fungal compounds in the seed cavity diffusate of resistant
genotypes and they confirmed that these diffusates strongly inhibited spore
germination of A. rabiei, Alternaria brassicae, and Colletotrichum falcatum.
25
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Chapter 2. Leterature Review
2.8.3 Chemical control
2.8.3.1 Seed treatment
Among the various factors that affect chickpea seed health, the most
important is the seed-borne fungus A. rabiei, though it is very important to
cultivate the healthy seeds by producing disease-free seeds or treating them
with the effective fungicides. The infected seeds with A. rabiei, in addition to
the production of infected seedlings, reduce their germination and the survive
ones are less vigorous, with poorly developed root systems and weak
stems(Ilyas, 1984). There were many agents and chemicals used for seed
treatment. Sattar(1933)was probably the first to achieve success in eradicating
the seed-borne A. rabiei by immersing the infected seed in 0.5% copper
sulfate solution, and also Kaiser et al.,(1973) found that the incidence of
blight in chickpea seedlings was greatly reduced, and it’s emergence
markedly had increased, when infected seeds were treated with certain
chemicals, especially with the systemic benzimedazoles (benomyl) and
thiobendazole (TBZ), they reported that treatment of seed with different
fungicides before planting did not protect the foliage of seedlings against
infection when the seedlings were artificially inoculated 2-3 weeks after
emergence. According to Kaiser(1973), the incidence of Ascochyta blight in
chickpea seedlings in Iran was reduced more than 80% and it’s emergence
increased to 45% when inoculated seeds treated with Benlate or
thiobendazole. Reddy(1980) had reported that A. rabiei eradicated in naturally
infected chickpea seeds by using systemic fungicide Calixin-M, used alone or
combining it with Benomyl. Maden (1983) who also discovered seed
treatment with Thiram (80% WP) + Benomyl (50% WP) in a 1:1 mix at 6
g/kg seed prevented disease transmission and increased emergence by
reducing damping-off. The seeds that are treated with Tecto, Benlate, Derosal,
26
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
and Baytan at rate of 3g/kg seed, gave the lowest percentage of infection, but
none of the chemicals completely eradicated the seed-borne infection(Saleem,
1984) also, Vitavax and Benlate suggested by(Merchant, 1984) for seed
treatment. In laboratory tests, Malik(1984b) identified effective fungicides,
namely Calixin-M, Benlate, Captan, and Tecto-60 as seed dresses. Also in
laboratory tests, the incidence of seed-borne A. rabiei reduced from 45% in
untreated to 0% in treated seeds by using Benomyl and thiobendazole
(TBZ)(Kaiser & Hannan, 1988). However, in a study in Iran, Sharafeh and
Banihashimi (1992) found that seed treatment with various fungicides had no
sufficient effect on infection rate or yield when there were high levels of soil
infestation by the pathogen. Nevertheless, Kaiser et al.,(1994) suggested
Captan or combination of Captan and Benomyl of seed treatment in Palouse
region, California, USA, for cultivation chickpea seeds in disease-free areas,
and according to Wiese et al.,(1995), specific formulations of Metalaxyl,
Captan, thiobendazole, and Benomyl are currently registered to apply on
chickpea seed. These fungicides significantly reduce, but may not eliminate
A. rabiei from seed. While Ambarder and Singh (1995) stated that the best
control of A. rabiei of grown chickpea achieved by seed treatment with
carbendazim + Thiram. Moreover, in greenhouse tests, Benomyl, Vencent,
and Tecto eradicated the disease transmission to seedlings (Al-Taee, 1997).
2.8.3.2 Control by foliar application
Since Ascochyta blight was the most devastating disease of chickpea
and spreads very quickly under favorable conditions, foliar spray is
especially needed to produce healthy seeds and in this aspect various
inorganic and organic spray fungicides have been reported to be effective
and reduce losses. Lukashevich (1958a) obtained good Control of chickpea
blight by spraying 3% aqueous sulfur at 500-600 lit/ha and increased yield
from 31 to 82.8%. The organic fungicides also proved to be effective in the
27
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
control of gram blight. Vir and Grewal(1974b) found that captan was
effective at 1kg/ 400 lit. water. According to Schwarts et al.,(1979) the best
control obtained by spraying Mancozeb every 7-14 days and applications
before flowering ensured high seed quantity and quality but the persistence
of conditions favorable for disease after flowering does not remove the
possibility of infected seeds. At ICARDA, the spraying with Bravo 500
(Chlorothalonil) at weekly intervals gave complete protection to a highly
susceptible cultivar under severe artificial epiphytotic condition(Reddy,
1984a). Tufail (1984) found Tilt and Daconil were effective in checking the
disease by affecting spore germination and periodic spraying is required to
control the disease by reducing the inoculum level. Ilyas and Bashir(1983)
used various systemic organic fungicides and found that most effective spray
fungicides in reducing disease rating were Tilt and TBZ, while the most
effective fungicides in reducing percent pod infection and percent disease
seeds were Tilt, TBZ, and Benomyl, but for seed production Reddy(1984d)
suggested spraying with Bravo at 7-10 day intervals, will prevent infection
in plots where blight may develop. Bashir and Ilyas(1984) had show that the
combinations
of
Benlate(benomyl)
with
Karathane(dinocap),
Daconil(chlorothalonil) or Dithane M-45(Mancozeb) caused the greatest
reduction in growth of the pathogen in culture, while in field trials, Gaur and
Singh(1985) found that Dithionon, Chlorothalonil, Captafol, and Captan
gave the best control of A. rabiei on chickpea.
Invitro studies, according to Abdou et al.,(1991a) spraying with 10
fungicides revealed that Benomyl(as Benlate), Tridemorph(as Calixin) and
thiobendazole inhibited A. rabiei at 10 PPM. Thiram prevented fungal
growth at 100 PPM while Chlorothalonil(as Bravo carb) and Zineb
prevented growth at 1000 PPM, but In vivo studies, Carboxin(as Vitavax) +
Captan and Mancozeb(as Dithane M-45) gave the lowest disease index.
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Chapter 2. Leterature Review
Also, Inam ul Haq et al.,(1995) found that A. rabiei was most sensitive to
Propiconazole(as Tilt), Penconazole(as Topas C-50) and sulfur(as Thiovit)
in vitro. Propiconazole and Penconazole completely inhibited the growth at
5 and 50µg/ml, respectively. The least effective fungicides tested were
Chlorothalonil(as Daconil) and Copper(as Cuprocaffaro). Mancozeb +
Metalaxyl(as Ridomil) and Propineb(as Antracol) displayed intermediate
effectiveness, after 28 day of incubation at 20±2oC. Chlorothalonil and
Propineb were most effective in reducing disease rating and increasing yield
in the field. Rauf et al.,(1996) observed that Chlorothalonil(as Daconil) and
Captan significantly reduced chickpea blight severity and increased yields
by 183.2 and 180.3%, respectively. On the other hand, according to TraperoCasas et al.,(1998), thiobendazole was effective in preventing the
development of the sexual stage of A. rabiei on naturally infected chickpea
debris.
Number of applications done by Islam et al.,(1999) and they observed that the
single or double application at 0.2% of Tilt, Daconil, Score-250 and Topas C50 suppressed the rate of gram blight disease development compared to
untreated control. None of the fungicides applied once or twice completely
inhibited the symptom development. Score-250 was the most effective
fungicide followed by Daconil, Tilt and Topas C-50 in the order, whether
applied singly or twice. Nevertheless, foliar applications of fungicides for
control of A. rabiei are impractical and uneconomical(Saleem, 1984; Nene &
Reddy, 1987) because more than six fungicide applications per season are
needed to control the disease(Reddy & Singh, 1983; Reddy & Singh, 1990c)
and most of contact type , making them less useful for application during
rains(Reddy et al., 1992), but the information obtained can be utilized in the
seed production programme(Saleem, 1984). In this aspect, Wiese et al.,(1995)
who argued that protecting green foliage during pod fill by spraying with
29
MSc. Thesis – Qasim Marzani
Chapter 2. Leterature Review
effective fungicides, is very important especially if blight symptoms appear
and increase in incidence and severity.
30
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
Chapter 3 MATERIALS ANDMETHODS
3.1 Materials
3.1.1 Instruments and equipment
The following Instruments and Equipment were used in this study:
Items
origin
-Autoclave
PriorClave, England.
-Sensitive balance
Sartorius, Germany.
-Blender
Mammonlex, Taiwan.
-Compound microscope
Olumpys, Japan.
-Cooled incubator
BDH, England.
-Disecting microscope
Olympus, Japan.
-Dry air oven
Memmert, Germany.
-Haemocytometer
Neubauer, England.
-Hood
local market.
-Magnetic stirrer
IKA-COMBIMAG RCT,Germany.
-Micrometer
Olympus, Japan.
-Petri plates 15 x 90mm
Pyrex, England
-Photograph camera(Digital)
Sony-Mavica, Japan.
-Photograph camera(normal)
Zinnet, Russia.
-Photomicroscope
Leitz, Germany.
-Refrigerator
Arcalik, Turkey.
-Water bath
HAKKE, Germany.
-Water distiller
GFL, Germany.
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Chapter 3. Materials and Methods
3.1.2 Culture media
The following culture media were used in this study:
3.1.2.1 Chickpea Seed Meal Dextrose Agar(CSMDA)
For the isolation and multiplication of A. rabiei, chickpea seed meal
dextrose agar(CSMDA) was used(Reddy, 1984c). This medium was prepared
by boiling 40g of chickpea seed meal in 400ml of distilled water for 20min.,
then the extract filtered with one layer of cheesecloth. Twenty gram of
dextrose and 18g of agar were dissolved separately in 400ml of distilled water
then mixed with the seed filtrate and finally additional distilled water was
added to bring the total volume to 1 liter and autoclaved for 15 min. under
121oC and 15psi. To avoid bacterial contamination, the antibiotic
streptomycin sulfate was added with shaking to the medium just before
solidification and pouring. One ml of streptomycin [1g(745 units/mg) in
750ml sterilized distilled water(S.D.W.)] is added to each 10ml of medium to
bring the concentration of streptomycin in the medium to 100 units/ml.
3.1.2.2 Water Agar(WA)
This culture medium is prepared from agar powder and distilled water
at the rate of 2% of agar and autoclaved as mentioned before and used for
preparing of single spore isolates.
3.2 Methods
3.2.1 Field survey
To record the incidence and distribution of Ascochyta blight of chickpea,
surveys were conducted during March, April, and May, 2002. The surveys
covered five representative chickpea growing areas in Erbil province (figure
3-1).
32
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
Figure(3-1): Locations of field survey for Ascochyta blight of chickpea in
Erbil province during 2002 season.
33
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Chapter 3. Materials and Methods
These included Qushtapa, Ainkawa, Shorish(Degala), Harir, and SoranDiana. In each location, two fields were selected, one of them was early
sown(Middle of February) and the other was late sown(Middle of March). In
each selected field, the percentage of disease incidence was calculated at
random, counting 20 plant/field using the crossing diameter method, and for
counting degree of infection, 20 other chickpea plants were randomly selected
and labeled in each field, by using 0-5 evaluation scale(Porta-Puglia et al.,
1996) which modified from 0 to 4 scale established by Vir and Grewal(
1974a):
0= no visible lesions.
1= a few small(up to 5mm2 ) lesions on stem and/or foliage.
2= superficial stem lesions exceeding 5mm2 and absence of stem girdling.
3= deep and extensive stem lesions, stem girdling that can cause breakage on
no more than one branch.
4= deep and extensive girdling stem lesions, causing breakage on more than
one branch followed by extensive wilting.
5= plant killed.
The average of individual records were classified as follows:
0-2.5 resistant; >2.5=susceptible
For counting disease severity this equation was used(Reddy, 1984b):
Disease severity
Sum of (No. of plants in each infetion group Respective degree of infection)
Total No. of plants/replication the highest degree of infection
For indicating sources of primary inoculum and evaluating disease
incidence and severity in these fields, information about presence of infected
chickpea debris, source of cultivated seed, seed treatment, and plant density
were collected.
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Chapter 3. Materials and Methods
3.2.2 Isolation and identification of the pathogen
3.2.2.1 From infected plants (Single spore method)
The pathogen was isolated from leaflets, stems, and pods collected during the
surveys of the fields. Following the Udupa et al.,(1998) method, and under
aseptic conditions, the individual lesions on the plant were cut by sterilized
sharp knife into small pieces of 1cm, leaving a small healthy portion on either
side, then the pieces were washed thoroughly in sterilized distilled water and
surface-sterilized in 0.5% sodium hypochlorite(Naocl) solution for 10 min..
After re-washing with sterilized distilled water(once), the pieces were placed
in 70% alcohol for 30 sec., then rinsed three times in sterilized distilled water
followed by drying on filter paper, placed on water agar(2% agar) plates and
incubated at 20oC, subsequently(after 2-4 days ) a small sector for growing
mycelium from a single lesion was transferred to CSMDA plates. Single
spores of isolates were subsequently obtained from each single-lesion as
follows: stock cultures of the fungus were immersed in sterilized distilled
water and shacked gently to release spores from the pycnidia. The resulting
spore suspension diluted to a concentration of 3.5x10 5 spores/ml-1 using
haemocytometer, dispersed on water agar plates, and incubated at 20oC to
induce germination. After 24h, a block of agar 20x20mm transferred to
microscopic slide and dissected into smaller squares(1.5 x 1.5mm) aseptically
using a multi-blade razor knife. Squares with single germinating spores were
identified microscopically and transferred to fresh CSMDA culture medium.
3.2.2.2 From chickpea debris (Dilution plate method)
After collecting the infected plant debris from different locations of Erbil
province, they were cut into small pieces of 1cm, then washed with tap water
for 0.5h then surface sterilized with 1% of Naocl for 2 min. After washing
them three times with sterilized distilled water, the pieces were dried on filter
35
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
paper and placed on CSMDA plates and incubated at 20 oC. Fungal growth
observed after 5-7 days of incubation, then a small pit of mycelial culture was
picked up and transferred to CSMDA plates until complete isolation and
purification. For preparing single-spore isolates, the culture that containing
mature pycnidia, mixed with sterilized distilled water for one minute by using
a slow speed electric blender already sterilized with 70% ethanol, and then
filtered through a three layer of cheesecloth. Spore suspension is placed in a
tube of 10ml, from which a clean sterilized pipette is used to transfer 1ml of
this dilution to a tube with 9ml sterilized water. A fresh sterilized pipette is
used to mix this dilution and transfer 1ml of it to another tube containing 9ml
sterilized water. This process continued for as many dilutions as required and
in the final dilution 1ml of spore suspension is dispersed on petri plates
contained CSMDA and incubated at 20 oC. After 10 days, when single spore
colonies appeared, a piece of mycelium was taken from each colony
separately and transferred to fresh CSMDA media and then the isolates were
differentiated based on morphological and cultural characteristics.
3.2.2.3 From chickpea seeds
Following the incubation agar method(Anonymous, 1993), seeds of three
cultivars, namely Local landrace(Harir), Marakishi(from Erbil Agriculture
Research Center) and Shami(from Harir sub-district), were tested for seed
infection with A. rabiei. These cultivars were sown in last season under
conditions of Erbil Province. The percentage of infection was counted by
taking 200 seed randomly from each cultivar, then the seeds were placed in a
cheesecloth and were washed with flowed tap water for 5 min., then were
surface sterilized with 1% of Naocl for 3 min.. After that, washed three times
with sterilized distilled water then placed on CSMDA plates at the rate of 10
seed/plate, and then incubated at 20 oC. After incubation period for 15 days,
the plates were checked for detection of A. rabiei, then the number of colonies
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MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
were counted in each plate and finally the percentage of infection was
determined.
3.2.3 Pathogenicity tests
For testing the pathogenicity(Koch’s postulate) of the fungus A. rabiei, the
experiment was conducted in a greenhouse belongs to department of biology
in the college of science. Seeds of the susceptible kabuli type Shami(ILC
1929) and the desi type Harir were planted in 20cm diameter pots containing
3.5kg of sandy soil sterilized with 1% formalin. The seeds were planted at the
rate of 10seed/pot and in 2.5cm deep using Complete Randomized
Design(CRD) with four replicates. For inoculation of plants, a spore
suspension of mixed isolates grown on CSMDA media, was prepared by
mixing the 15day old cultures with sterilized distilled water for one minute by
using a slow speed electric blender already sterilized with 70% ethanol. The
spore suspension was filtered with three layers of sterilized cheesecloth and
adjusted to 1 x 106 spore ml-1(Jamil et al., 2000) with the aid of a
haemocytometer. Tween 20(one drop/100ml) was added to the spore
suspension as a wetting agent, then the two week old plants were inoculated
with the spore suspension until run-off by using the 500ml hand sprayer. The
pots were rotated during the inoculation procedure to ensure an equal
distribution of the spore suspension on the plants. Four other pots for each
treatment were left without inoculation, sprayed by the same manner with
sterilized distilled water and kept as a control. Then the pots have been
covered with transparent plastic bags for 72h to maintain high moisture, after
that they were left in greenhouse(temperature=17-25oC, RH=70-90%) until
the appearance of the disease symptoms. The appearance date of symptoms
on the leaves and stems was determined, color and size of symptoms were
described for each cultivar, and after 15 days from inoculation, degree of
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Chapter 3. Materials and Methods
infection was also recorded by using the 0-5 scale as described in section 3-21.
3.2.4 Study of virulence of A. rabiei isolates
During the surveys of the disease in 2002 season in Erbil province, 13
isolates of A. rabiei were isolated from chickpea plants, three of these isolates
were in Qushtapa, four in Harir, and two isolates in both of Ainkawa, Shorish,
and Soran. These isolates were different from one another in cultural
characteristics, number of pycnydia, sporulation, and growth rate(Qureshi,
1984b; Dolar and Gurcan, 1992). Each isolate was separately grown on
CSMDA media. The plant material chickpea(Cicer arietinum) accessions
Ghab-2(ILC
3279)
and
Ghab-3[resistant(R)],
Harir
and
IPA-
510[Tolerant(T)], and Shami(ILC 1929)[susceptible(S)] were selected as a
host differential set for testing the virulence of A. rabiei isolates. Then seeds
of these cultivars which surface disinfected with Naocl(active CL 2%) for 15
min and then rinsed with tap water(Porta-Puglia et al, 1996), were grown in
greenhouse(17-25oC, RH 70-90 % ) in 12cm diameter pots contained 1.5kg of
sterilized sandy soil and in 2.5 cm deep in a rate of 5 seed/pot with two
replicates. The pathogenicity of each isolate was tested individually on
infection these five differential cultivars, and for this purpose, the spore
suspension was prepared from each isolate and adjusted to 1 x 10 6 spore ml-1
using haemocytometer. Tween 20(one drop/100ml) was added to the spore
suspension as a wetting agent, then the two weeks old plants inoculated with
the spore suspension, and during the inoculation procedure, the pots were
rotated to ensure an equal distribution of the spore suspension on the plants.
After inoculation, each two sets that sprayed with one isolate, were covered
individually with transparent plastic bag for 72h. Disease development was
recorded 15 days after inoculation on each individual plant according to 0-5
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MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
scale of Porta-Puglia et al., (1996), then the isolates were grouped to the
pathotypes according to their virulence.
3.2.5 Study of the host range
For determining host specificity of A. rabiei, an experiment was
conducted in greenhouse with 19 legume plant species(Table 3-1). After
treating with Raxil(3gm/kg seed), seeds of these host plants had been planted
in 20cm plastic pots containing 3.5kg sterilized sandy soil at the rate of 10
seed/pot and in three replicates. The two week old seedlings were inoculated
by spraying technique until run-off with the mixed of A. rabiei spore
suspension adjusted to 1 x 10 6 spore ml-1. The spore suspension is a mixture
of isolates originated from different locations of Erbil province. Tween
20(one drop/100ml) was added to the spore suspension as a wetting agent,
and during the inoculation procedure, the pots were rotated to ensure an equal
distribution of the spore suspension on the plants. After inoculation, the plants
were covered with transparent plastic bags to offer high humidity for 72h,
then the pots were left uncovered in greenhouse until the appearance of
symptoms.
3.2.6 Production of sexual stage (Pseudothcia) in the
laboratory
3.2.6.1 On naturally infected chickpea debris
The infected plant debris, which contained visible pycnidia in the lesions, has
been collected from different locations of Erbil province through the last
season. The debris is cut into pieces of 2-4 cm long, after sterilizing the
surface with 1% of Naocl for three minutes, the stem pieces were washed and
placed on the 10 cm diameter pots filled with sterilized sandy soil, having five
pores at the bottom to allow water arise within the soil to maintain high
39
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Chapter 3. Materials and Methods
moisture, then the pots were placed inside a plastic cabinet containing 5cm
height of water.
Table (3-1): Host range of some legume plant species used in reaction of
chickpea blight caused by A. rabiei.
Ser.
Host name
Scientific name
1
Alfalfa
Medicago sativa
2
Bean
Phaseolus vulgaris L.
3
Birds foot trefoil
Lotus conniculatus
4
Broad bean
Vicia vapae L.
5
Chickpea
Cicer arietinum L.
6
Clover
Trifolium alexandrium
7
Common vetch
Vicia sativa L.
8
Cowpea
Vigna sinensis L.
9
Grass pea
Lathyrus annuas L.
10
Gray
Vicia merbella
11
Gray
Vicia marbonensis
12
Gray
Vicia mariana
13
Green gram
Phaseolus aureus
14
Indian pea
Lathyrus sativa L.
15
Lentil
Lens culinaris
16
Medic
Medicago polymorpha
17
Peas
Pisum sativum
18
Soya bean
Glycine max
19
Wild chickpea(black seed)
Cicer spp.
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Chapter 3. Materials and Methods
The cabinet was kept closed with a polyethylene bag to maintain high
humidity, placed in an incubator at 20oC for four days and transferred to an
incubator at 5oC. Subsequently, the samples were weekly taken for examining
formation of sexual stage and determining the period needed of immature and
mature pseudothecia formation. Asci were considered mature if they
contained fully formed ascospores. The immature and mature pseudothecia,
ascus, and ascospore were described, hence the dimensions of 100
pseudothecia, asci, and ascospores were measured by using the micrometer
and then compared to the previous measurements.
3.2.6.2 On artificially inoculated chickpea debris
The procedure used to produce the sexual stage was similar to that used by
Armstrong et al.,(2001). Dried chickpea stem pieces(6 to 8cm) were
autoclaved and immersed for 1h in the mixed spore suspension adjusted to 1 x
106 spore ml-1. After 1h, stem segments were drained and placed in sterilized
petri plates containing 10 discs of sterilized filter paper moistened with 15ml
of sterilized distilled water. The dishes were incubated for 24h at 20 oC and
then transferred to 10oC for 7 weeks. Sterilized distilled water was added
weekly to maintain high humidity in the dishes. The petri plates were not
sealed or placed in an airtight container. Subsequently, the formation of
sexual stage was monitored weekly examined using a compound microscope.
The period needed for maturation of sexual stage was determined,
pseudothecia, asci and ascospores were described as well as the dimensions of
100 pseudothecia, asci, and ascospores were measured. Discharge of
ascospores from the stem materials was induced according to Navas-Cortes et
al.,( 1998a) as follows: the stem segments were attached to petri plates lid
using a block of water agar and then suspended over water agar in the bottom
of the petriplates. After 1-2 days the agar surface was examined for Didymella
rabiei ascospores, the discharged ascospore was picked up with agar blocks to
41
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
the CSMDA medium for producing cultures for the purpose of inoculating
chickpea plants to ensure that the sexual stage belongs to A. rabiei.
3.2.7 Effect of natural environmental factors on the
disease
The field experiment was conducted in the Girdarasha fields(5km South of
Erbil) for studying the effects of environmental factors on the disease
development. The cultivars, Harir, Shami, and Marakishi(from Erbil
Agricultural Center), Ghab-2, and Ghab-3 (from Sulaimani Agriculture
research Center), and Dijla and IPA-510(from IPA center for Agricultural
researches, Mosul), were planted in the middle of December in 2001-2002
season by using Randomized Complete Block Design(RCBD) with three
replicates, each one consisted of seven treatments and each treatment
contained 5 rows of 5m length. The inter and intra-row spacing followed were
30 and 10cm, respectively(Reddy & Singh, 1990a). The plots were artificially
inoculated with infected chickpea debris collected from previous season by
scattering the infected debris at the rate of 50g/plot. Two weeks after their
emergence, the plants were re-inoculated with a mixtures of spore suspension
adjusted to 1 x 106 spore ml-1 to ensure uniform infection in epiphytotic form.
In each treatment, 25 plants were labeled at a rate of 5 plant/row. Degree of
infection was weekly determined(by using a 0-5 scale) for each cultivar
starting from the appearance of the disease symptoms until the end of May,
when the environmental conditions for blight development became
unfavorable at the end of the season. The daily temperature, relative
humidity(RH), rainfall and leaf wetness period were recorded starting from
the emergence of the seedlings until the end of the season.
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Chapter 3. Materials and Methods
3.2.8 Effect of the disease on yield quantity and quality
For detecting yield loss caused by Ascochyta blight, a chickpea field
planted with the cultivar Shami(S) in Qushtapa region, was selected .The area
of the field which was planted in the middle of February in 2002 season, was
approximately 40 donum and have the plant density at 12.6 plant/m2. For
monitoring the incidence and development of the disease, regular surveys
were carried out. The percentage of infection was reached to 95%, in the end
of the season, therefore various degrees of infection were recorded on an
individual plant that ranged from 0 to 5(figure 4-2 in section 4-1) using 0-5
scale. At the end of the season, twenty plant were taken randomly from each
group of infection using a crossing diameter method, then the number of pod,
seed, seed yield/plant, weight of 100 seed, seed yield, biological
yield(seeds+straw), and percentage of yield loss for each degree of infection
were counted. Similarly, the average degree of infection and the average loss
of the field were counted. The following equation was used for counting yield
loss(Reddy & Singh, 1990a):
YL%
YHP YDP
100
YHP
Where:
YL= Yield loss
YHP= Yield of healthy plants
YDP= Yield of diseased plants
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Chapter 3. Materials and Methods
3.2.9 Control Methods
3.2.9.1 Control by changing date of sowing
The experiment was conducted in the college of Agriculture’s field in
Girdarasha. In this experiment, two sowing dates were followed, the first was
the middle of December(Winter sowing) and the second was at the beginning
of March(spring sowing). In each sowing date, seven cultivers were planted
which are Harir, Shami, Marakishi, Dijla, IPA -510, Ghab-2, and Ghab-3, in a
factorial RCBD with three replicates. Each one contained 14 treatments and
each treatment consisted of 5 rows in a length of 5m. The inter and the intra
row spacing were 30, 10cm, respectively, leaving 2m among the treatments
and 4m among the blocks. The treatments were artificially inoculated by
scattering infected chickpea debris, which collected from the previous season
in a rate of 50g/treatment. Two weeks after the emergence, the plants were reinoculated with spore suspension of mixed isolates adjusted to 1x 10 6 spore
ml-1 to ensure uniform infection in epiphytotic form. In each treatment, 25
plants were taken at the rate of 5plant/row for measuring degree of infection,
disease severity, percentage of stem infection, degree of stem infection, and
plant height. The biological yield and seed yield by harvesting internal rows,
weight of 100seed, harvest index, percentage of pod infection, and percentage
of seed infection were also measured. The percentage of seed infection was
measured by growing 200 seed from each treatment on CSMDA medium
following the incubation agar method(Anonymous, 1993), and for measuring
Harvest Index(HI), this formula was used(Hussain, 1984) :
HI Economic Yield 100
Bio log ical Yield
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Chapter 3. Materials and Methods
The following scale was used for evaluating degree of stem infection (AlTaee, 1997):
0= no infection.
1= simple spots on branches less than 2 mm.
2= spots 2-6mm long with no complete girdling.
3= big spots more than 6mm with complete girdling.
4= branches killed.
And for measuring severity of pod infection, the following scale was used(AlTaee, 1997):
0=no infection
1= 1-25% of pod surface infected
2= 26-50% of pod surface infected
3= 51% or more of pod surface infected
The results were statistically analyzed and differentiated by Duncan,s
Multiple Range Test(DMRT).
3.2.9.2 Screening chickpea germplasms
3.2.9.2.1 in the greenhouse
For determining the resistant cultivars of chickpea against Ascochyta
blight, the following cultivars were grown in greenhouse which was, Harir,
Shami, Marakishi, Dijla, IPA-510, Ghab2 and Ghab 3. Seeds of each cultivar
were planted in 2.5cm deep in 22cm diameter pots containing 3.5kg of
sterilized sandy soil at the rate of 10 seed/pot. The experiment was
implemented with CRD in four replicates. Two weeks after emergence, the
plants were inoculated with a spore suspension of mixed isolates adjusted
to1x 106 spore ml-1. The pots were rotated during the inoculation as usual, and
then the plants were covered with transparent plastic bags for 72h to obtain
high humidity for infection. Three weeks after the inoculation, the degree of
45
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
infection, percentage of stem infection, the degree and severity of stem
infection were counted as were mentioned previously.
The results were statistically analyzed and differentiated by DMRT.
3.2.9.3 In the field
The same cultivars above in greenhouse trials were planted under field
conditions in Girdarasha in 15 Dec. 2001 using RCBD with three replicates,
each one contained seven treatments and each treatment has 5 rows in a
length of 5m. The inter and intra-row spacing were 30 cm and 10 cm,
respectively. The treatments were inoculated with infected chickpea debris,
which collected from the previous season. Two weeks after the emergence,
the plants were re-inoculated by spraying with spore suspension of mixed
isolates adjusted to 1 x 106 spore ml-1, for the uniformity and insurance of
infection. Degree of infection percentage of stem infection, and it’s severity,
was measured before crop maturing when disease development was checked,
as mentioned in section 3-2-9-2-1. The results were statistically analyzed and
differentiated by DMRT.
3.2.9.4 Chemical control
Although the chemical control is impractical and uneconomical with
susceptible cultivars especially under epiphytotic conditions, but it can be
used for seed treatment for eradicating the disease from infected seeds,
similarly in disease free areas and for foliar application of producing diseasefree seeds. On the other hand, fungicides application can be use as an
alternative control measure for blight at least until resistant cultivars become
available.
46
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
3.2.9.4.1 Food poisoning technique for fungicides efficacy on growth
of A. rabiei
The effect of eight fungicides, which were used for seed treatment and
foliar application, were tested on growth of A. rabiei(Table 3-2) in vitro. Each
fungicide was mixed with warm medium(45 oC) just before pouring at the rate
of 100 mg of a.i./liter medium and then poured into 7cm diameter petri plates.
After solidification, the plates were inoculated in their centers with
0.4cm diameter discs of 15-day-old A. rabiei cultures that were grown on
CSMDA medium. The experiment was implemented by using complete
randomize design(CRD) in four replicates. Four other petri plates contained
CSMDA only, inoculated in the same manner used as control.
The results were calculated after two weeks of incubation at 20 oC by
measuring the average of the two crossing diameters for each colony and then
the percentage of the inhibition were counted according to the following
equation:
% of inhibition
Average diameter of the controlcolony - Average diameter of the treated colony
Average diameter of the controlcolony
X 100
The results were statistically analyzed and differentiated by DMRT.
3.2.9.4.2 Seed treatment
3.2.9.4.2.1 In Vitro studies
Seeds of cultivar Shami(S), which were visually shown to be infected, were
treated with the following fungicides: Benlate 50%, Raxil 2DS, Rovral,
Mancozep 80% and Dividened 3 %, at the rate of 3gm/kg seeds using CRD in
three replicates. The seeds were grown on 9cm diameter petri plates contained
CSMDA medium at the rate of 10 seed/petri plate and untreated infected
seeds were also grown in other three petri plates as control. After two weeks,
number of A. rabiei colonies and percentage of grown seeds were counted.
The results were statistically analyzed and differentiated by DMRT.
47
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
3.2.9.4.2.2 In Vivo studies
The same five fungicides in section 3-2-9-3-2-1 were tested in greenhouse by
planting treated infected seeds of the same cultivar(Shami) with the same
fungicides at the rate of 3g/kg seeds. The seeds were grown in 2.5cm deep in
22cm diameter pots containing 3.5kg sterilized sandy soil and at the rate of 10
seed/pot using Complete Randomized Design(CRD) in four replicates.
Table(3-2): Trade name, common name, and chemical structure for eight
fungicides used in seed treatment and foliar application against
Ascochyta blight(Meister et al., 2001).
Trade Name
Raxil
DS2
Rubigan
Common Name
Tebuconazol
Fenarimol
4%EC
Chemical structure
2(4chlorophenyl)ethyl(1,1,dimethyl
ethyl)-1H-1,2,4-triazole-1-ethanol.
-(2-chlorophenyl)--(4-chlorophenyl)
-5-pyrimidine-methanol.
1-[2-[4-(chlorophenoxy)
Dividend
Difenoconazole
30FS
2-chlorophenyl]-4-methyl-1,3-dioxon2-ylmethyl]-1H-1,2,4-triozole.
3-(3,5-dichlorophenyl)-N-
Rovral
Iprodione
50%WP
Domark
10EC
Benlate
50%WP
Mancozeb
80%WP
(1-methylethyl)-2,4-dioxo-1imidazolidine carboxamide.
Tetraconazole
Benzimidazole
Mancozeb
2-(2,4-dichlorophenyl)-3(1H-1,2,4-triozole-1-yl) propyl.
Methyl-1-(butylcarbamyl)-2benzimedazole carbamate.
Zinc
ion,
manganeseethyl-ene
bisdithiocarbamate.
48
MSc. Thesis – Qasim Marzani
Topas
Penconazole
100EC
Chapter 3. Materials and Methods
1-[2-(2,4-dichlorophenyl)pentyl]11,2,4-triozole.
Four other pots were grown with untreated infected seeds as control and four
pots grown with healthy seeds for measuring the effect of disease on the
percentage of germination. The pots were left in greenhouse until the
appearance of infection on the plants, then the percentage of germination,
germination speed and percentage of infected plants were counted.
The following equation was used for counting the speed of germination:
GerminatinSpeed
No. of germinatedseed in the 1st day ... No. of germinatedseed in the7th day
Total No. of seedlings
And this equation used for counting percentage of the Germination:
G % L 100
S
Where: G= percentage of germination.
L= No. of germinating seedlings.
S= total No. of seeds.
The results were statistically analyzed and tested by DMRT.
3.2.9.4.3 Foliar Application
3.2.9.4.3.1 In greenhouse
To determine the effect of fungicides on the Ascochyta blight on
chickpea plants, the following fungicides were selected for post infection
spray: Benlate 50 %WP, Raxil DS2, Rubigan 4%EC, Topas 100EC, and
Domark 10EC. Seeds of the susceptible cultivar, Shami, were planted in 12cm
diameter pots containing 1.5kg sterilized sandy soil and at the rate of 5
plant/pot. The two weeks old plants were inoculated with spore suspension of
49
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
mixed isolates adjusted to 1x10 6 spore ml-1 and then were covered with
transparent plastic bags for 72h, then the pots left in greenhouse until the
appearance of the disease. At the beginning of the appearance of the disease,
the plants were sprayed with the above mentioned fungicides and according to
the recommendations of the production companies which were 1g/liter for
both Benlate and Raxil, 1ml/liter for Rubigan, 0.5ml/liter for Topas, and
0.35ml/liter for Domark. The plants were sprayed until run-off and rotated
during the spraying in both inoculating and chemical spraying. The
experiment was implemented in a CRD in three replications. Three other pots
were sprayed with sterilized distilled water as control. After three weeks of
application, degree of infection using 0-5 evaluating scale, percentage of stem
infection, dry weight, and plant height were measured. The results were
analyzed statistically and differentiated by DMRT.
3.2.9.4.3.2 in the field
The foliar spray experiment was conduced in Girdarasha fields by
planting seeds of the susceptible cultivar Shami in middle of February 2002.
The experiment was designed as factorial RCBD with three replicates. The
levels of factor A included the following fungicides:
Raxil DS2
Tapas 100EC
Benlate 50 %WP
Rubigan 4%EC
Domark 10EC
,and levels of factor B included the following spraying times and dates :
b1=Two sprays, one after the appearance of disease symptoms and the other
after 14 days starting from the first spray.
50
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
b2=Three sprays, the first after appearance of disease symptoms, the second
after 10 days from the first spray, and the third spray after two weeks from the
second spray.
b3=Three sprays, the first after the appearance of the disease, the second and
the third spray were simultaneously with the rainfall and were implemented
before sunset.
In each block, one plot left without any spraying with fungicides as
control, sprayed with the water only. Each block contained 16 treatments and
each treatment consisted of 4 rows in a length of 5m, the inter and intra-row
spacing were 30 and 10 cm, respectively, while the inter and intra-block
spacing were 4 and 2m, respectively. The treatments(plots) were artificially
inoculated with infected chickpea debris which collected from the previous
season in a rate of 40g/plot . Two weeks after the emergence, the plants were
re-inoculated with a spore suspension adjusted to 1x 10 6 spores ml-1, for
uniformity and insurance of appearance the disease. At the beginning of the
appearance of the disease, the first spray(application) was implemented then
followed by other applications according to designed application times and
dates. In the end of the season, the percentage of infection, degree of
infection, percentage of pod infection, biological yield, seed yield, harvest
index(HI), and percentage of seed infection were measured. The results were
statistically analyzed and differentiated by DMRT.
51
MSc. Thesis – Qasim Marzani
Chapter 3. Materials and Methods
52
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
Chapter 4 Results and Discussions
4.1 Field survey
The results of the field survey in chickpea fields in Erbil province during
March, April, and May of 2002 season, revealed that the disease was present
in all locations including the place of survey and it appeared during March in
early sown(middle of February) fields in all locations. The highest percentage
and degree of infection during this month were 80% and 1.18, respectively, in
Ainkawa(Table 4-1) and this is probably due to suitable environmental
conditions during March in this area, or may be due to the high percentage of
infected seeds sown by farmer’s in that field, and the presence of chickpea
plant debris from previous season in adjacent the field. In this aspect, NavasCortes et al.,(1998b) reported that on debris of naturally infected chickpea,
ascospore maturation occurred mainly from late January to late March. The
lowest percentage and degree of infection during the same month were
recorded in Shorish, which were 10% and 0.1, respectively. Figure(4-1)
shows that the highest disease severity during March was in the early sown
field in Ainkawa which was 0.24, while the lowest one was 0.02 in Shorish.
On the other hand, table(4-1) also shows that there were no infection in the
late sown fields during this month except in Harir which infected in a rate of
5% and have a degree of 0.05, and this may be partially due to the endemic of
disease in this location where after harvesting, the farmers collect their crops
inside the fields and after threshing the seeds yield, they leave the chickpea
straws in the field which is considered as a source of primary inoculum and
from it the disease will initiate during next season. Another reason was a high
percentage of infection of farmers cultivated seeds which was 4% (Table 4-1).
53
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
These results agree with statements that of Kaiser et al.,(1994) whom
confirmed that both infected plant debris and infected seeds were the major
sources of disease incidence. In April, the disease was increased in all the five
locations, thus the infection in the early sown fields reached to 100% in
Ainkawa, Harir, and Soran.
Table(4-1): The percentage and degree of infection of Ascochyta blight on
chickpea plants of some fields during the survey 2002 in Erbil province.
Source of inoculum
April
May
March
April
May
Sowing
date
Degree of infection
March
Location
%of field infection
Plant
density
% of
infected (plant/ m2 )
seed
Early
sowing
None
1%
12.6*
25
90
95
0.25
2.5
2.83
Late
sowing
None
1%
7
0
65
85
0
Early
sowing
Adjacent
the field
6%
8.53
80
100
100 1.18 3.65 4.15
Late
sowing
Adjacent
the field
0%
5
0
50
75
0
0.82 1.23
Early
sowing
None
0%
8.53
10
30
40
0.1
0.35 0.45
Late
sowing
Adjacent
the field
1.50%
13.33
0
60
65
0
1.18 1.23
Early
sowing
Adjacent
the field
2%
17.4
30
100
100
0.3
Late
sowing
Inside the
field
4%
11.93
5
85
100 0.05 1.37 2.53
Early
sowing
Inside the
field
0.50%
15.13
35
100
100 0.35 3.62
4.4
Late
sowing
Adjacent
the field
3%
20.6
0
75
100
3.53
Chickpea
debris
Qushtapa
0.88 1.33
Ainkawa
Shorish
3.85
5
Harir
Soran
54
0
1.7
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
This rapid increase of disease attack in these locations might be due to the
presence of two sources of inoculum, which were infected seeds, and plant
Qushtap
Ainkawa
Shorish
Harir
Late
Early
Late
Early
Late
Early
Late.
Early
Late
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
March
April
May
Early
Disease severity
debris of the previous season in adjacent fields.
Soran
Locations and date of sowing
Figure (4-1): Severity of Ascochyta blight on chickpea in some fields of
Erbil province during 2002.
During April the disease appeared also in all late sown fields in all locations.
The highest percentage of infection in this sowing date (85%) was in Harir,
while the highest degree of infection and disease severity were in Soran which
were 1.70 (table 4-1) and 0.31(figure 4-1), respectively. The substantially
increased during May in both sowing dates keeping in mind that different
degrees of infection were observed (figure 4-2), therefore, in early sowing,
three high degree of infections have been recorded in Harir, Soran, and
Ainkawa which were 5.00, 4.40, and 4.15, respectively, and have disease
severities of 1.00, 0.83, 0.83, respectively. These three fields were completely
devastated by the blight disease and resulted with 100% yield loss, and have
been re-plowed. From Table (4-1) generally, we observe that the degrees of
infections in the late sowing fields were lower than the early ones. It can be
55
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
concluded that the farmers can protect their crops from the blight damage by
shifting the sowing date from early to the late spring, especially with the
susceptible cultivars such as Shami.
Figure(4-2): Different degrees of infection of Ascochyta blight on
chickpea plants during 2002 season(ranged from 0-5).
In contrast with the above view, the diverse results were observed in Shorish
that the percentage and degree of infection as well as disease severity in late
sowing trials were greater than early sowing(Table 4-1 and figure 4-1), and
this is possibly due to sowing infected seeds, presence of infected plant debris
in adjacent of the late sown field, but the main factor of this result probably a
high chickpea plant density of this late sown field in comparison with the
early sown field which permit to success the transmission of the inoculum. It
should be remembered that the survey was conducted in fields were no
fungicides used for seed treatment.
4.2 Disease symptoms in the field
The symptoms of Ascochyta blight on chickpea appeared on all foliar parts of
the plants, and the severely infected fields appeared as a blighted area from
56
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
long distances(figure 4-3b) if it is compared with the healthy ones (figure 43a).
a
d
h
b
c
e
f
i
g
j
Figure (4-3): Ascochyta blight symptoms on chickpea
Where: a: healthy field, b: infected field, c: broken stem, d: infected stem,
e: girdled stem , f: healthy leaf , g: infected leaf , h: infected seeds ,
i: healthy seeds , j: infected pod
The circular or elongated brown spots appeared on the leaves(figure 4-3g),
and when the petioles get infection it become girdled and whole leaf will
57
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
break down. The spots usually contain black dots which were asexual fruiting
bodies(pycnidia) of the fungus that can be observed inside the spots in
concentric rings. On the stems, the brownish elongated lesions have been
observed with brown to black pycnidia(figure 4-3d), and the infected stems
usually girdled(figure 4-3e) causing the break of the stem(figure 4-3c). On the
pods the lesions were usually circular with dark margins and obvious
arranged pycnidia(figure 4-3j). When pod infection become severe, the
fungus will attack the seeds which become shriveled and black spots usually
observed on the seed coats(figure 4-3h).The above descriptions of the
symptoms on the plant parts is in consistent with the descriptions that of
Guzman et al.,(1995), who observed symptoms on the plants in the fields
included tan to brownish lesions on leaves, stems, and pods, with brown to
black pycnidia immersed in the host tissue and arranged in concentric rings
within the lesions.
4.3 Isolation and identification of A. rabiei
4.3.1 From infected plants
The direct isolation of pathogen from infected chickpea plants(leaves, stems,
and pods) with the aid of cultural characteristics, growth rate, descriptions and
dimensions of pycnidia and pycniospores, as well as confirmation of Koch’s
postulates, confirmed that the causal organism was A. rabiei(Pass.) Labr.. All
isolates of the fungus were able to grow and form colonies on CSMDA
medium at 20oC in a slow growth rate(figure 4-4a), therefore, the average of
colony diameter was 23mm after two weeks of incubation. The color of
colony in the beginning was white then shortly turned to dark because of the
formation of pycnidia. The fungus have a dark, septate and branched
mycilium(figure 4-4c). The pycnidia were circular or ovular in shape, yellow
58
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
to brown in color, and has the average diameters of 152.43 x 181.84µm(figure
4-4b).
b
a
c
d
e
Figure(4-4): The pathogen Ascochyta rabiei
a: appearance of the vegetative growth(the culture)
b: asexual fruiting bodies(pycnidia)-200X
c: vegetative body(the mycelium)-400X
d: pycnidiospores out of the ostiole of pycnidia-200X
e: pycnidiospores(conidia)-400X.
After maturation of pycnidia, the pycnidiospores (conidia) observed out in the
ostiol(figure 4-4d), which were bright and hyaline and usually non-septate
59
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
(figure 4-4e), have the average width and length of 4.37and 9.19µm,
respectively, and these dimensions approximately are similar to those of A.
rabieii
which
identified
and
described
by
Kovachevski(1936b),
Punithalingam and Holliday(1972), and Al-Taee(1997).
4.3.2 From chickpea debris
The results of isolation from infected chickpea debris revealed that the
fungus was A. rabieii, which grown in a pure culture and its description was
entirely similar to that described in section 4-3-1. In this aspect the present
results strongly agree with statements of Wiese et al.,(1995) whom confirmed
that the pathogen will survive in infested crop residues and in infected seeds
from season to another.
4.3.3 From chickpea seeds
The results of isolation of A. rabieii from seeds of cultivars Shami, Local
landrace(Harir),and Marakshi, showed that the percentage of seed infection of
these cultivars were at the rate of 4, 1, and 3%, respectively. The results were
approximately similar to that of Grewal(1982) who noted that seed infection
ranged from 3 to 17.5% for the desi varieties and from 7.5 to 60.5% for the
kabuli ones. The results also confirm that the disease was seed transmission
by which the disease will spread to new areas. It should be remembered that
the descriptions of the isolated fungus from seeds were similar to that
identified in section 4-3-1.
4.4 Pathogenicity tests
The results of pahogenicity tests(Koch’s postulates) revealed that A.
rabiei isolates could infect both cultivars Shami and Harir at 100% infection,
but they differ in degree of infection which were 3.7 and 2.3, respectively(on
60
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
0-5 scale). Both chickpea genotypes showed symptoms on both leaves and
stems. The first symptoms were appeared on the Shami in the 5th day and on
the cultivar Harir in the 6th day. The symptoms on the young leaves of cultivar
Shami at the beginning of the appearance were bright to straw-colored, and
this is similar to the descriptions reported by Reddy(1984a) who stated that
small, round, white necrotic specks appeared on newly formed leaves of
susceptible cultivars. The symptoms on the cultivar Harir were brown-bright
with dark margins. These symptoms on both cultivars resembled to those
shown in the field. The pycnidia was brown to black dots appeared within the
lesions on Shami and Harir cultivars after 3 and 4 days of the appearance of
symptoms, respectively. The results are in a agreement with that of Hohl et
al.,(1990) they confirmed that the development of leaf spots and fungal
pycnidia could be observed 6-8 day post infection. The results also near to
that of Trapero-Casas and Kaiser(1992b) who stated that in constant
temperature of 20oC, the minimum incubation and latent periods were 4.5 and
5.5 days, respectively.
4.5 Study of virulence of A. rabiei isolates
The results in the table (4-2) shows the virulence reactions of different
isolates on different cultivars. The most virulent one was isolate 2 that most
selected cultivars were susceptible toward this isolate except Ghab-2 and
Ghab-3. The least virulent isolates were 1, 8, 9, 10, and 11, that most selected
cultivars were resistant toward these isolates except the cultivar ILC 1929
which was the only cultivar susceptible toward these isolates. Meanwhile, the
moderate virulent isolates were 3, 4, 5, 6, 7, 12, and 13. We concluded that
both cultivars, Ghab-2 and Ghab-3, were resistant against all isolates, while
the cultivar Shami(ILC1929) was susceptible toward all isolates. Therefore,
the results confirm variability in virulence among isolates of different
61
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
locations and these differences confirm the presence of races or pathotypes of
the pathogenic fungus. The results apply to the statements that of Porta-Puglia
et al.,(1996) who could distinguish three groups of isolates in Italy when he
used 41 Italian isolates which differentiated by using 13 chickpea genotypes,
and with that of
Table (4-2): Reaction of 13 different isolates of A. rabiei on five selected
cultivars of chickpea in Erbil province.
No. of
Ghab-2
Shami
Location
Harir
IPA-510 Ghab-3
isolate (ILC3927)
(ILC1929)
Qushtapa
Ainkawa
Shorish
Harir
Soran
1
2
3
4
5
6
7
8
9
10
11
12
13
1.10*R
1.65 R
1.15 R
1.60 R
1.10 R
1.00 R
1.00 R
1.00 R
1.10 R
1.00 R
1.20 R
1.95 R
1.15 R
1.55 R
2.55 S
1.80 R
1.55 R
1.65 R
1.15 R
2.30 R
1.85 R
1.75 R
1.40 R
1.30 R
2.30 R
1.95 R
2.20 R
3.00 S
3.05 S
3.00 S
2.95 S
2.60 S
2.85 S
2.05 R
2.00 R
1.75 R
2.10 R
3.05 S
2.70 S
1.51 R
1.50 R
1.30 R
1.40 R
1.00 R
1.00 R
1.50 R
1.30 R
1.00 R
1.10 R
1.75 R
1.45 R
1.25 R
4.50 S
4.90 S
5.00 S
5.00 S
4.90 S
5.00 S
3.60 S
4.50 S
4.00 S
4.60 S
4.50 S
4.70 S
4.00 S
R= Resistant(degree of infection 0.0-2.5).
S= Susceptible(degree of infection >2.5).
* Each value was means of two replicates
Table(4-3): Disease assessment in the form of resistant(R) and
susceptible(S) reaction shown by 13 isolates of A. rabiei on five selected
chickpea cultivars.
Ghab2
Shami
Pathotype
Harir
IPA-510 Ghab-3
(ILC3279)
(ILC1929)
R
R
R
R
S
A
R
R
S
R
S
B
R
S
S
R
S
C
A= Isolates: 1, 8, 9, 10, and 11, B= Isolates: 3, 4, 5, 6, 7, 12, and 13, C= isolate 2.
62
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
Noorolahi(2000) when he distinguished two races among 400 isolates
collected from different provinces of Iran. Another confirmation of our results
was those of Jamil et al.,(2000) that three pathotypes were selected within 130
isolates in Pakistan. In Iraq, four races were distinguished by Al-Taee(1997)
among 31 isolates collected from different locations of Ninavah province by
using 12 differential cultivars. Table(4-3) shows the presence of three
pathotypes in Erbil province which were A, B, and C. The most aggressive
pathotype was C, which could infect most of selected cultivars except
cultivars Ghab-2, and Ghab-3, while the least aggressive one was A that most
selected cultivars were resistant against this pathotype except cultivar Shami.
The results revealed the presence of the three pathotypes (A, B, and C) in
Qushtapa, pathotype B only in both Ainkawa, Shorish and Soran, and
pathotype A in Harir. The presence of more than one pathotype in Qushtapa
my be because that in this location, many different cultivars will be sown.
Several reasons, such as the increase of chickpea-growing area in this location
may contribute to extension of the variability of virulence. It should be
remembered that the fungus is heterothallic (Kaiser et al., 1998) and the
results of production of sexual stage in section 4-7, confirm the presence of
both mating types in the region that was the cause of this variation in
virulence among pathotypes, therefore more variation could be expected.
4.6 The host range
4.6.1 Fitness costs
The results of artificial inoculation of 19 legume plant species with mixture of
spore suspension, revealed that Ascochyta blight symptoms appeared on
leaves and stems of chickpea crop(Cicer arietinum L.) and wild chickpea
(Cicer spp.) after one week from inoculation. Simple disease symptoms were
63
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
also appeared on the leaves only and without forming pycnidia on 7 other
legume species (table 4-4), and this result were confirmed by re-isolating the
pathogen from these infected plant species.
Table (4-4): Host range of some legume plant species infected by A.
rabiei.
Host
name
Scientific name
Type of symptoms
Dark lesions on the leaves and
stems
Wild
Dark lesions on the leaves and
Cicer spp.
stems
chickpea
Straw colored lesions on the
Alfalfa
Medicago sativa L.
leaves only
Indian
Lathyrus sativa L. Dark lesions on the leaves only
pea
Gray
Vicia merbella
Grey lesions on the leaves only
Small yellow spots on the
Gray
Vicia mariana
leaves only
Yellow lesions on the leaves
Gray
Vicia marbonensis
only
Grass
Complete drying of some
Lathyrus annus L.
compound leaves
pea
Small yellow spots on the
lentil
Lens culinaris
leaves only
Chickpea Cicer arietinum L.
Presence of
pycnidia
Yes
Yes
No
No
No
No
No
No
No
This result is in agreement with Kaiser (1991) who observed reddish, brown
lesions on alfalfa plants, but he claimed that pycnidia also appeared in
necrotic tissues. These differences in disease symptoms may be due to the
variability in virulence of A. rabiei races. We agree also with finding of
Montorsi et al.,(1992), who stated that A. rabiei isolated from barseem clover
seeds, was purified and multiplied and inoculating on barseem and chickpea
plants, the symptoms appeared after 10-12 day on both hosts. Our results were
in contrast with that of Al-Taee(1997) who showed that A. rabiei could infect
the genus Cicer only, and this is probably due to the differences in virulence
between Erbil isolates with that of Ninavah. On the other hand, Kaiser(1973)
64
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
and Gaur and Singh(1993a) could isolate the pathogen from cowpea and
phaseolus, likewise Khan et al.,(1999b) also could re-isolate A. rabiei from
chickpea and 4 common bean species after artificial inoculating of 20 plant
species with an isolate of A. rabiei obtained originally from chickpea in south
Australia. Wiese et al.,(1995) asserted that hosts other than chickpea, such as
pea, alfalfa, certain other legumes, and some weeds may be rarely and weakly
attacked by the pathogen. Such infections may remain latent or invisible, or
may result in mild disease symptoms. These alternative hosts, however, may
play a role in the local survival of blight fungus.
4.7 Production of sexual stage
4.7.1 On naturally infected chickpea debris
The results of production of sexual stage under laboratory conditions at
5oC2, revealed that the immature sexual fruiting bodies(pseudothecia) were
observed at the third week of examination as a shine-black dots on the surface
of chickpea debris(figure 4-5a), and it is similar to asexual fruiting bodies
(pycnidia) in the shape and color(figure 4-5b), but it can be easily
distinguished by the use of microscope which revealed that the presence of
conidia inside the fruiting bodies confirm that the fruit body was pycnidia,
while immature sexual bodies did not contain conidia, except fungal tissues
were found inside these bodies. The mature sexual fruit bodies were formed at
8th week(figure 4-5c) and this was confirmed by the presence of the mature
asci(figure 4-5d) which contained 8 bi-celled ascospores(figure 4-5e). Similar
conclusions were made by AI-Taee(1997), who stated that the mature sexual
fruiting bodies were formed at 9th week, however, this difference possibly was
due to variability of races or may be because of the differences in incubation
temperatures and other environmental conditions. According to Navas-Cortes
et al.,(1998b), on debris of naturally infected chickpea, ascospore maturation
65
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
occurred mainly from late January to late March and maximum ascospore
discharge occurred 2-4 weeks after ascospore maturation. They reported also
that asci and ascospores per pseudothecium were much higher in cooler
locations.
b
a
c
d
e
Figure(4-5): The sexual stage(pseudothecia) of Mycosphaerella rabiei :
a: pseudothecia as black dots on the surface of chickpea stem(51.2X)
b: immature pseudothecia(200X)
c: mature pseudothecia with asci(200X)
d: ascus contain 8 bi-celled ascospores(500X)
e: ascospores(1250X)
66
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
Navas-Cortes et al.,(1995) stated that on debris left on the soil surface under
the natural conditions, the fungus could rapidly colonized the tissues forming
abundant pseudothecia and pycnidia, likewise under controlled conditions in
the laboratory, Didymella rabiei extensively colonized, moreover they added
that the incubation temperature was the principal factor associated with the
production of conidia and ascospores in particular. The sexual fruiting bodies,
which were circular or oval, have the diameter ranged 130.6-168 μm and
contained unlimited numbers of club-shaped asci and each ascus contained 8
bi-celled, unequal, and hyaline ascospores. The ascus dimensions were 47.592.5μm × 9.75-15µm with the average 68.24µm×12.53 µm, and ascospore
size was 10-16.25µm×5-9.25µm. These dimensions are approximate to the
measurements reported by AL-Taee(1997) and Armstrong et al.,(2001).
The presence of sexual stage confirm that both mating types, Mating
type І and ІІ were present in Kurdistan region because the fungus was
heterothallic and needs both Mating types to the formation of sexual
stage(Kaiser et al., 1998). The formation of sexual stage under high RH and
low temperature indicates that when both two conditions prevail in winter, the
sexual stage formation can be predict which may serve as primary
inoculum(Akem, 1999) and contributes to long distance spread of the disease
by air-borne ascospores(Kaiser and Kusmenuglu, 1997). Trapero-Casas et
al.,(1996) also reported that the airborne ascospores of Didymella rabiei is
the major primary inoculum of Ascochyta blight epiphytotics in chickpea crop
in southern Spain. Another confirmation were made by Kaiser et al.,(1994)
that in USA an epiphytotic form observed in Palous region in 1987 when
much of the chickpea crop were grown in isolated field experiments and were
devastated by Ascochyta blight. They also stated that these isolated fields
were infected by the blight pathogen at distances more than 8 km from the
nearest chickpea planting. According to Trapero-Casas et al.,(1996), the
67
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
ascospores were trapped mostly from the beginning of January to the late
February, this period coincided with that of maturity of pseudothecia on the
chickpea debris, therefore Autumn-winter sowings of chickpea were exposed
longer to ascospore inoculum than the traditional spring sowings.
4.7.2 On artificially inoculated chickpea debris
The results of the weekly examination of inoculated chickpea stems
under 10oC and RH of 100%, showed the presence of mature pseudothecia as
a black shine dots on the surface of chickpea stems in the 7 th week(figure 45a). These findings are similar to that of Armstrong et al.,(2001) who stated
that mature ascospores can be discharged at 7th week of inoculation. In this
aspect Navas-Cortes et al.,(1998a), stated that Didymella rabiei grew
saprophytically on pieces of artificially and naturally infected chickpea stem
debris under artificial incubation conditions, formed pseudothecia and
pycnidia and they added that the pseudothecia matured at 10 oC and constant
100% RH, or at 5 and 10oC and alternating 100% and 34% RH and for
temperatures higher than 10oC or RH lower than 100%, pseudothecia either
did not form ascospores, or ascospores did not mature and their content
degenerated.
It should be remember that the descriptions and measurements of
pseudothecia, asci, and ascospores on artificially inoculated chickpea debris
were similar to those described in section 4-7-1. The inoculation of chickpea
plants with cultures originated from ascospores, resulted in infection of
chickpea plants and the symptoms that appeared were similar to those of A.
rabiei. These results confirm that the sexual stage was belonged to the blight
pathogen A. rabiei.
68
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
4.8 Study of the effect of environmental factors on the
disease
The results obtained from the weekly recording of infection of the seven
cultivars, revealed that the disease symptoms appeared in the last week of
January(after 44 days of planting) on all grown cultivars(figure 4-6). The
symptoms appeared when the weekly average of maximum temperature and
average RH were 10.29oC and 70.07%, respectively, with the total amount of
weekly precipitation and leaf wetness period of 47.2mm and 16h. Our results
are more similar with that of Jhorar et al.,(1998b) whom noted that the
disease severity increased with wetness duration, approximately of 18h and
theyconfirmed, that the average RH and maximum temperature, during the
crop season, were good indicators of the epiphytotic conditions in the crop
field. Another confirmation of our results was those of Trapero-Casas and
Kaiser(1992b) who stated that the optimum temperature for infection and
development of Ascochyta blight of chickpea, in 2 week old seedlings in
controlled environment studies was 20 oC, and at this temperature 7.6 and 17h
of wetness were required for the pathogen to cause significant light and severe
infection, respectively. They also confirmed that the lower and upper limits
for infection and disease development were <5 and about 30oC. On the other
hand, the results of Ketelaer et al.,(1988), support our finding, however, they
reported that the monthly average temperature of at least 8 oC and monthly
precipitation of at least 40mm is necessary of an epiphytotic of A. rabiei.
In the third week of the appearance of the disease, the percentage of
infection reached to 100% on both cultivars, Shami and Marakishi, and in the
6th week on IPA-510, Ghab-2, and Ghab-3, but on the cultivar Harir, 100%
infection occurred in the 9th week.
69
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
We concluded that all cultivars were infected, but they varied in the
degree of infection(disease severity). The disease continued for its
acceleration during March and April when suitable environmental
conditions(low temperature and high humidity) were prevailed. These results
strongly agree with statements given by Kaiser(1995) that the disease spreads
rapidly when cool and wet conditions prevail.
In the first week of May(in the 15th week of infection), the disease was
checked, because during this week of May the average maximum temperature
was 28.5 and the average RH was 36.43. In agreement with the present
results, Reddy and Singh(1990b) stated that the weekly maximum
temperature more than 25oC and weekly mean RH less than 60% slowed
progress of blight disease.
The most rapid increase of degree of infection was observed on both
Shami and Marakishi cultivars which occurred at 5th week from infection
which reached to 4.2 and 4.7, respectively(on the 0-5 scale), then finally they
completely devastated. This rapid progress of the disease possibly was due to
the susceptibility of these two cultivars. The cultivars, Harir, Dijla, Ghab-2,
and Ghab-3 remained resistant until the end of the season and their degree of
infection reached to 2.46, 1.62, 1.95, and 2.22, respectively. On the other
hand the cultivar IPA-510, remained resistant until the 9th week, while its
disease severity is increased rapidly within few weeks during flowering and
pre-flowering stages, then finally the degree of infection reached to 3.29.
We observed that despite of prevail of epiphytotic conditions, the cultivars
Harir, Dijla, Ghab-2, and Ghab-3 were resistant against isolates of Erbil
province, but the cultivars Shami, Marakishi, and IPA-510 were susceptible
under the same above conditions.
70
Rainfall(mm)
Leaf wetness period(h)
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
40
35
30
25
20
15
10
5
0
55
50
45
40
35
30
25
20
15
10
5
90
0
80
70
60
Relative
Humidity(%)
50
40
30
20
10
0
40
Temperature(C
o
)
35
30
25
20
15
10
5
0
5
Harir
Shami
Marakishi
Dijla
IPA-510
Ghab-2
Ghab-3
Degree of infection
4.5
4
3.5
3
2.5
2
1.5
1
27 May
20 May
13 May
29 Apr.
22 Apr.
15 Apr.
8 Apr.
1 Apr.
25 Mar.
18 Mar.
11 Mar.
4 Mar.
25 Feb.
18 Feb.
11 Feb.
4 Feb.
28 Jan.
21 Jan.
14 Jan.
7 Jan.
31 Dec.
0
6 May
0.5
Figure (4-6): effect of temperature, RH, rainfall and leaf wetness period
on the degree of infection on seven chickpea cultivars.
71
MSc. Thesis – Qasim Marzani
Chapter 4. Results and Discussions
4.9 Effect of the disease on seed yield quantity and
quality
Table (4-5) which shows the effect of the blight disease on seed yield quantity
and quality, and how the disease causes serious crop losses with increasing of
degree of infection, in comparison with the yield of healthy plants. The
lowest yield loss of 16.04% occurred when the plants have one degree of
infection(on 0-5 scale),while the losses became 98.64% with the degree of
5.The results are similar to some extent with those of Acikgoz et
al.,(1994)were they stated that sometimes Ascochyta blight may cause 100%
yield losses. This table also shows that the number of pods, seeds, and seed
yield/plant were decreased with the increasing degree of infection. The weight
of 100 seed, seed yield/ha, and biological yield/ha were also decreased with
increasing degree of infection. The yield loss was also counted which
decreased with increasing degree of infection. Finally, the average degree of
infection of the field was counted with counting of all above-mentioned
variables. We noted that the average yield loss of the field was 60.18% and
this result is agreed to some extent with that of Gaur and Singh(1993b) who
stated that losses of 77-84% were recorded in Sri Ganganagar, India.
4.10 Control methods
4.10.1 Effect of sowing date on the
4.10.1.1 degree of infection
The
sowing
date
affected
significantly
on
the
degree
of
infection(Table4-6). Thus, the Spring sowing date reduced the degree of
infection at the rate of 28.25%.
72
Chapter 3. Results and Discussions
Table(4-5): Effect of Ascochyta blight on seed yield, quantity and quality.
% of each
degree of
infection
No. of
pod/plant
No. of
seed/plant
Seed
Yield/plant
(g)
Weight
of 100
seed
Seed
Yield
(kg/ha)
Biological
Yield
(kg/ha)
Yield
loss
(kg/ha)
% of
Yield
loss
5
37.35*
31.50
15.40
51.84
1925.00
4116.37
.000
0.00
5
27.45
26.20
12.93
51.74
1616.25
3674.18
308.75
16.04
25
24.45
23.60
12.13
51.21
1516.25
3605.43
408.75
21.23
35
8.25
7.00
3.54
49.01
442.50
1424.12
1482.50
77.01
4
25
4.85
3.85
1.73
43.36
216.25
840.56
.170875
88.77
5
5
1.00
0.70
0.21
31.28
26.25
172.37
1898.75
98.64
12.23
6.13
47.54
766.38
2008.09
1158.63
60.18
Degree
of infection
0
1
2
3
Average
2.8
13.50
73
Chapter 3. Results and Discussions
This difference between the two sowing dates in the degree of infection
possibly is due to reason that the Winter sown plants grown under favorable
conditions for disease development during Winter and early Spring(Figures 46), so the pathogen has more time to develop the disease in comparison with
the Spring sown. The present results strongly agree with the results obtained
by Singh and Reddy(1990), who stated that the farmers save their chickpea
crops from Ascochyta blight by follow-through the Spring sowing. The same
results were confirmed by Reddy and Singh(1990b) who reported that the
disease development will be slow at the temperatures more than 25oC.
Table(4-6) shows that there were significant differences between
cultivars in this regard, both culltivars Shami and Marakishi were the most
susceptible which have degree of infections of 4.69 and 4.78, respectively,
with no differences between them, while both cultivars have significant
differences with the other cultivars.
From the results of interaction between the sowing dates and cultivars,
we observed significant differences between the two sowing dates in all
cultivars. Although, the degrees of infection of both cultivars, Shami and
Marakishi, in Spring sowing were less than that of Winter sown, which were
4.37 and 4.55, respectively, but their degrees were also very high. This is may
be because of the susceptibility of both cultivars during early stages of growth
in which the disease developed in a rapid form within few days under suitable
environmental conditions.
4.10.1.2 Disease severity
Table(4-6) shows that there were significant differences between both
sowing dates in disease severity, therefore the spring sowing caused reduction
in disease severity at 27.87 % than the winter sowing. There were also
significant differences between cultivars in disease severity. Two high disease
74
Chapter 3. Results and Discussions
severities were recorded on both cultivars Shami and Masrakishi, which were
0.94 and 0.95, respectively, with no significant differences between the two
cultivars, but have them with the other cultivars. There were also no
significant differences between Dijla and Ghab-2, while Dijla have the lowest
disease severity.
From the table(4-6), we observed the effect of interaction between
sowing dates and cultivars. The disease severity in Spring sowing plants were
less than that of Winter sowing with significant differences between both
sowing dates in all cultivars.
4.10.1.3 Percentage of stem infection
The results revealed that there were significant differences in percentage of
stem infection between Winter and Spring sowing plants. The percentage of
stem infection was reduced in Spring sown at 34.68%(Table 4-6). On the
other hand, the significant differences were shown among cultivars in related
to percentage of stem infection. The two susceptible cultivars, Shami and
Marakishi have a high percentage of stem infection, which were 86.19 and
89.61%, respectively, with significant differences between them. The
cultivars Dijla, Ghab-2 and Ghab-3 have very low percentage of stem
infection which can be negligible which are 1.28, 1.98 and 2.74%,
respectively, with no significant difference among them(Table 4-6). This low
levels of stem infection possibly is due to the resistance of these cultivars, in
this aspect the present results strongly agree with the statements of Singh et
al.,(1992b) who stated that the cultivar Ghab2(ILC 3279) was resistant to A.
rabiei and with statements of AL-Taee(1997) that Dijla was resistant to the
blight pathogen.
The results of interaction between sowing dates and cultivars shows that there
were significant differences between Winter and Spring sowing in cultivars
75
Chapter 3. Results and Discussions
Harir, Shami, Marakishi, IPA-510, and Ghab-3. While there were no
significant differences between the two sowing dates in cultivars Dijla and
Ghab-2.
4.10.1.4 Degree of stem infection
From Table(4-6) which represents the effect of sowing date on degree
of stem infection, it seems that there are significant differences between
Winter and Spring sown, thus, the degree of stem infection was reduced in
Spring sowing at 27.55% in comparison with that of Winter. There were also
significant differences among all cultivars in the degree of stem infection. The
highest degree was 3.91 in cultivar Shami while the lowest one was 0.75 in
Dijla. There were also significant differences in interaction between sowing
dates and cultivars except in cultivars Shami and Marakishi that were no
significant differences observed.
4.10.1.5 Percentage of pod infection
There were significant differences between Winter and Spring sowing
in term of percentage of pod infection. From Table(4-6), we observed that
there were no infection on pods in Spring sowing. This is probably because of
the presence of unfavorable environmental
conditions for disease
development during pod formation stage in the first half of May(figures 4-6).
The highest percentage of pod infection was 3.42% on the cultivar Harir and
have significant differences with other cultivars. Cultivar Ghab-3 infected in a
percentage of 1.58% with significant differences with the other cultivars,
while there were no significant differences among cultivars Shami, Marakishi,
Dijla, IPA-510, and Ghab-2.
The results of interaction between sowing dates and cultivars, revealed
that there were significant differences between the two sowing dates in
76
Chapter 3. Results and Discussions
cultivars Harir and Ghab-3, while there were no significant differences in the
rest. The Cultivars, Shami and Marakishi did not reach to the pod formation
stage in Winter sown because the plants were killed by the pathogen in the
early stages of their ages.
Inspite of the significant differences between Winter and Spring sowing in
percentage of infected chickpea pods, the percentage of infection were very
low in Winter sowing and this may be due to the presence of short period of
suitable environmental conditions for disease development during pod
formation stage in May.
4.10.1.6 Pod infection severity
From Table(4-6), we observed that there were significant differences
between Winter and Spring sowing in pod infection severity. The cultivar
Harir has significant differences with the other cultivars, while all other
cultivars did not differ with one another significantly. In the interaction of
sowing dates and cultivars, the significant differences observed only in two
cultivars, which were Harir and Ghab-3.
4.10.1.7 Percentage of seed infection
The results in this study showed that there were significant differences
in percentage of seed infection between Winter and Spring sowing(table 4-6).
The seeds of Spring sowing were entirely have no infection, this is possibly
due to the fact that there were no pod infections in Spring sowing. Both
cultivars Harir and Ghab-3 have significant differences with the other
cultivars. The highest percentage of seed infection occurred in seeds of
cultivar Harir, which was 1.25% and this probably because this cultivar was
susceptible during pod formation stage.
77
Chapter 3. Results and Discussions
From Table(4-6) which also represents the interaction between sowing
dates and cultivars, we observed that there were significant differences in
percentage of seed infection in cultivars Harir, IPA-510, and Ghab-3. The
results showed that the percentage of seed infection was lower than the
percentage of pod infection in the same cultivars, this is may be due to
superficially infection(less severity) of pods of these cultivars(table 4-6), or
may be due to the fact that pod infections not always cause seed infection.
4.10.1.8 Weight of 100 seed
In present work, the results in the table(4-7) showed that there were
significant differences between both sowing dates in weight of 100 seed,
hence the Winter sowing has more weight of 100 seed. The seed weight was
reduced in Spring sowing in a rate of 12.45%.
There were significant differences in weight of 100 seed among
cultivars Harir, Shami, Marakishi, Dijla, and IPA-510, while both Ghab-2 and
Ghab-3 did not differ with each other significantly, but have significant
differences with all other cultivars. The results of interaction between sowing
dates and cultivars revealed that there were significant differences between
both sowing dates in weight of 100 seed in cultivars Harir, IPA-510, Ghab-2,
and Ghab-3, while the cultivar Dijla did not differ significantly in weight of
100 seed between the two sowing dates. Both Shami and Marakishi not
yielded during Winter sowing and their seeds of Spring sowing have less
weight of 100 seed because the plants of these cultivars severely affected by
the disease in Spring. In this aspect, Carvalho et al.,(1999), recorded severe
yield losses for chickpea fields grown under favorable condition due to reduce
of weight of 100 seed.
78
Chapter 3. Results and Discussions
Pod infection
severity
% of seed
infection
W.S. S.S.
Harir
2.46d* 1.56f
2.01c
0.49e 0.31i
0.40 c
7.49 e 1.73 fg
4.61 d
2.46 c 1.66 e
2.10 d
6.83 a 0.00 c
3.42 a
0.0166 a 0.0000 c
0.0083 a
2.50a 0.00d
1.25 a
Shami
5.00a
4.37b
4.69a
1.00a 0.88c
0.94 a
100 a 72.37c
86.19b
4.00 a 3.82 a
3.91 a
(-)**
0.00 c
0.00 c
(-)
0.0000 c
0.0000 b
(-)
0.00d
0.00 c
Marakishi
5.00a
4.55b
4.78a
1.00a 0.91c
0.95 a
100 a 79.22b
89.61a
4.00 a 3.71 a
3.86 b
(-)
0.00 c
0.00 c
(-)
0.0000 c
0.0000 b
(-)
0.00d
0.00 c
Dijla
1.62f
1.04g
1.33e
0.32hi 0.21j
0.27 e
2.29 f 0.27 g
1.28 e
1.24 f
0.26 g
0.75 g
0.00 c 0.00 c
0.00 c
0.0000 c 0.0000 c
0.0000 b
0.00d 0.00d
0.00 c
IPA-510
3.29c
1.83ef
2.56b
0.66d 0.37gh
0.51 b
19.22d 1.10 g
10.16c
2.87 b 2.15 cd
2.51 c
0.83 c 0.00 c
0.42 c
0.0010 c 0.0000 c
0.0005 b
0.50c 0.00d
0.25 c
Ghab-2
1.95e
1.02g
1.49de
0.39fg 0.20j
0.29 e
3.60 ef 0.35 g
1.98 de
1.89 de 0.60 g
1.25 f
0.16 c 0.00 c
0.08 c
0.0002 c 0.0000 c
0.0001 b
0.00d 0.00d
0.00 c
Ghab-3
2.22d
1.13g
1.68d
0.44ef 0.22j
0.33 d
5.22 ef 0.26 g
2.74 de
2.17 cd 1.27 f
1.72 e
3.16 b 0.00 c
1.58 b
0.0043 b 0.0000 c
0.0022 b
1.50b 0.00d
0.75 b
Average
3.08a
2.21b
W.S.
S.S.
0.61a 0.44b
W.S.
S.S.
33.97a 22.19b
W.S.
S.S.
W.S.
S.S.
2.20 a 0.00 b
2.66 a 1.92 b
*Values followed by a common letter are not significantly different at P=0.05, according to Duncan,s multiple range test
**Not counted because the plants not reached to the yield stage.
W.S.= Winter sowing.
S.S.= Spring sowing.
Note: each value represents means of three replicates.
79
W.S.
S.S.
0.0044 a 0.0000 b
W.S.
S.S.
0.90a 0.00b
Average
% of pod
infection
Average
Degree of
stem infection
Average
% of stem
infection
Average
Disease severity
Average
Degree of
infection
Average
The
cultivar
Average
Table(4-6):The effect of date of sowing on the Ascochyta blight development of seven chickpea cultivars.
Chapter 3. Results and Discussions
4.10.1.9 Seed yield
The results in table(4-7) showed that the seed yield was greatly reduced by
shifting the date of sowing from Winter to Spring, therefore inspite of the
avoidance of A. rabiei damages to some extent in Spring sowing, the seed
yield was reduced at the rate of 432.04 kg/ha and in percentage of 36.5%.
In this aspect, statements of Ilyads(1998) confirm our results that the yield in
Winter sowing were consistently higher between 26-330% than the Spring
sowing. Similar conclusions reported by many workers, like Singh(1990) who
reported that a minimum of 500kg/ha additional yield can be obtained in
Winter sowing. Hawtin and Singh(1984) noted that the yield of chickpea can
be increased by sowing it in Winter(early December) provided that Ascochyta
blight is controlled.
Table(4-7) shows There were significant differences between cultivars
in seed yield, but the cultivar IPA-510 graded first which have a seed yield of
1525.14kg/ha, then followed by cultivars Ghab-3(1482.38), Harir(1387.92),
Dijla(1316.14), and Ghab-2(1043.02kg/ha), while both Shami and Marakishi
have too low(negligible) seed yields which were 1.50 and 16.50 kg/ha
,respectively. This is because that the plants of both cultivars were killed by
the pathogen in Winter and those of Spring sowing were severely infected and
resulted of too low yields. The results of interaction between sowing dates
and cultivars in Table(4-7) showed that there were significant differences
between the two sowing dates in all cultivars except in cultivar Shami.
4.10.1.10 Biological yield
The present results in Table (4-7) shows
significant differences
between Winter and Spring sowing in biological yield. The Winter sowing
proved its superiority in increasing biological yield and have more biological
yield of 1609.73kg/ha than the Spring sowing. There were also significant
80
Chapter 3. Results and Discussions
differences between the cultivars in the biological yield, therefore the highest
biological yield which was 4357.66kg/ha obtained in Harir cultivar, followed
by IPA-510, Ghab-3, Dijla, and Ghab-2, respectively. The results of
interaction between sowing dates and cultivars revealed that there were
significant differences between both seasons in all cultivars.
4.10.1.11 Harvest Index (HI)
The results of the present study in Table(4-7) shows that there were
significant differences between sowing dates in the harvest index(HI), though
the Spring sowing have more HI at the rate of 16.84% than the Winter
sowing. This unexpected result is probably due to that both cultivars Shami
and Marakishi not yielded during Winter sowing(section 4-10-1-9). Similar
conclusions were made by AL-Taee(1997) who confirmed that sowing in the
middle of March were superior in HI than sowing in middle of December and
have more HI in a rate of 16.20%. The cultivar Ghab-3 has most HI and this
is because of the high seed yield of this cultivar.
The results of interaction between sowing dates and cultivars showed
that there were significant differences between two sowing dates in cultivars
Harir, Marakishi, Dijla, IPA-510, and Ghab-3, but there were no significant
differences between the two sowing dates in cultivars Shami and Ghab-2.
4.10.1.12 Plant height
From Table(4-7), we observed significant differences between Winter and
Spring sown plants in term of plant height, therefore the Winter sowing have
the average plant height more than the Spring sowing at the rate of 27%. The
results of Singh et al.,(1997) confirm our results, however, they showed that
Winter sowing plants were taller than those sown in Spring, permitting
harvesting by combines. Table(4-7) also shows that the cultivars Ghab-2
81
Chapter 3. Results and Discussions
came first in plant height and differed significantly with all other cultivars.
The cultivars Harir, Dijla, and Ghab-3 also have reasonable heights with no
differences among them, but differed significantly with the other cultivars.
Both cultivars Shami, and Marakishi have too low heights, this is
possibly due to the fact that the plants of the two cultivars severely infected
during both sowing dates.
The results of interaction between sowing dates and cultivars showed that
there were significant differences in plant height between Winter and Spring
sowing in cultivars, Harir, Dijla, IPA-510, Ghab-2, and Ghab-3, while both
cultivars Shami, and Marakishi did not differ significantly.
4.10.2 Screening Chickpea germplasm
4.10.2.1 In the greenhouse
The results of artificially inoculation for seven cultivars in greenhouse
(table-8) confirmed that both cultivars Shami and Marakishi were susceptible
to isolates of A. rabiei which were from Erbil province, but the cultivars
Harir, Dijla, Ghab-2, and Ghab-3, were resistant to the blight pathogen. The
cultivar IPA-510, inspite of recording the degree of infection of 2.21 which
considered resistant, but it was relatively high degree that could be considered
as a tolerant cultivar.
From table (4-8) we observed that the low degrees of stem infections were
also recorded in resistant cultivars, Harir, Dijla, and Ghab-2 with no
significant differences among them.
The cultivar Marakishi has highest
degree of stem infection, then the cultivar Shami comes in the second order.
The lower percentage of stem infection were recorded in resistant cultivars
Harir, Dijla, Ghab-2 and Ghab-3 with no significant differences among
them(table4-8).
82
Chapter 3. Results and Discussions
Harir
30.68 b*
28.95 c
29.82 c
1682.92 c
Shami
(-)**
31.00 b
31.00 b
0.00
l
3.00
l
Marakishi
(-)
17.64 f
17.64 f
0.00
l
33.00
k
Dijla
26.23 d
26.26 d
26.25 d
1357.28 e
IPA-510
38.92 a
31.35 b
35.14 a
Ghab-2
26.10 d
22.61 e
Ghab-3
25.02 d
22.34 e
Average
29.39 a
25.74 b
1092.92 h
W.S.
S.S.
Harvest index
W.S.
S.S.
Plant height(cm)
W.S.
S.S.
Average
S.S.
W.S.
Biological yield(kg/ha)
Average
S.S.
Seed yield(kg/ha)
Average
W.S.
Average
Weight of 100
seed (g)
The
cultivar
Average
Table(4-7):The effect of date of sowing on the some field traits of seven chickpea cultivars.
1387.92 c
5560.32 b
3155.00 f
4357.66 a
30.27 g
34.66 d
32.47 c
56.93 b
44.20 cd
50.57 b
1.50
g
280.48 l
842.32 k
561.40 g
0.00 k
0.36 k
0.18 f
5.87 f
9.73 f
7.80 d
16.5
f
280.56 l
965.32 j
622.94 f
0.00 k
3.42 j
1.71 e
4.60 f
10.40 f
7.50 d
1275.00 f
1316.14 d
4970.80 d
2977.92 g
3974.36 d
27.31 h
42.81 b
35.06 b
62.60 ab
40.40 d
51.50 b
1850.28 b
1200.00 g
1525.14 a
5030.28 c
3610.28 e
4320.28 b
36.78 c
33.24 e
35.01 b
48.13 c
31.27 e
39.70 c
24.36 e
1474.48 d
611.56 j
1043.02 e
5537.92 b
2318.00 i
3928.00 e
26.63 i
26.38 i
26.50 d
65.93 a
45.13 cd
55.53 a
23.68 e
1919.76 a
1045.00 i
1482.38 b
5849.68 a
2373.04 h
4111.36 c
32.82 e
44.04 a
38.43 a
58.67 b
40.00 d
49.33 b
1183.53 a
751.50 b
3930.00 a
2320.27 b
21.97 b
26.42 a
43.25 a
31.59 b
*Values followed by a common letter are not significantly different at P=0.05,according to Duncans multiple range test
**Not counted because the plants not reached to the yield stage.
W.S.= Winter sowing.
S.S.= Spring sowing.
83
Note: each value represents means of three replicates.
Chapter 3. Results and Discussions
The susceptible cultivars, Shami and Marakishi have high percentages
of stem infection. The cultivar Marakishi ranks first in stem infection severity
then followed by cultivar Shami. There were low stem infection severities in
resistant cultivars Harir, Dijla, Ghab-2, and Ghab-3 and they did not differ
significantly with one another, while the cultivar IPA-510 did not differ
significantly with susceptible cultivars, Shami and Marakishi.
We concluded that under greenhouse conditions, the cultivars Harir,
Dijla, Ghab-2, and Ghab-3 were resistant(figure 4-7) and have low degree of
stem infection, percentage of stem infection, and stem infection severities,
while the cultivar IPA-510 was tolerant to the disease, but both cultivars
Shami and Marakishi were susceptible.
4-10-2-2-In the field
The results of variability in resistance to A. rabiei in the field confirmed
that the cultivars Shami and Marakishi were most susceptible in the degree of
infection, degree of stem infection, percentage of stem infection, and stem
infection severity, followed by IPA-510 which was moderately susceptible
(Table 4-8), but the cultivar Harir was moderately resistant(or tolerant) in the
above mentioned variables, while the cultivars Dijla, Ghab-2, and Ghab-3
proved their resistance against the disease and have the lowest degrees of
infection, degree, percentage, and severity of stem infection. These results
confirm the previous ones that were obtained in the greenhouse. In this
aspect, Singh(1990) stated that Ghab-2(ILC 3279) released in Syria as a
resistant cultivar to A. rabiei.
84
Chapter 3. Results and Discussions
Table(4-8): Screening germplasm against Ascochyta blight in both greenhouse and the field.
Degree of infection*
The cultivar
Greenhouse
Degree of stem
infection**
Field
Greenhouse
Field
Stem infection severity
% of stem infection
Greenhouse
cd
Field
Greenhouse
Field
7.49 c
0.37 c
0.61 bc
53.08 b
100 a
0.74 a
1.00 a
4.00 a
62.55 a
100 a
0.79 a
1.00 a
1.58 cd
1.24 d
6.00
2.25 e
0.38 c
0.32 e
3.29 b
2.43 bc
2.75 b
10.41 c
19.22 b
0.60 ab
0.67 b
1.23 c
1.98 d
1.05 de
1.89 d
3.18
d
3.6
0.26 c
0.45 d
1.58 c
2.20 d
1.86 c
2.17 c
5.11
cd
5.22 cd
0.43 bc
0.54 c
Harir
1.65 c***
2.46 c
1.49 ce
2.46 bc
5.68
Shami
2.54 ab
5.00 a
2.95 ab
4.00 a
Marakishi
2.80 a
5.00 a
3.25 a
Dijla
1.53 c
1.62 e
IPA-510
2.21 b
Ghab-2
Ghab-3
cd
de
* On the 0-5 scale
** On the 0-4 scale
***Values within a column followed by a common letter are not significantly different at P=0.05, according to Duncans multiple range test.
Note: Each value is means of three replicate
85
Chapter 3. Results and Discussions
The present results of the cultivar IPA-510 were in contrast with results that
of Al-Taee(1997), however, he claimed that the cultivar was resistant to A.
rabiei in Mosul province in both greenhouse and the field. This disagreement
possibly was due to the variability in virulence between isolates from Mosul
and Erbil, or may refer to that the resistance in the cultivar IPA-510 did not
last long, which might be break down and became susceptible. In this aspect
Singh (1984) stated that in Pakistan the resistant cultivar F8 became
susceptible after some years of planting. Confirming to our results, Reddy et
al.,(1983), had reported that the resistant genotypes in Syria became
susceptible when were grown in Pakistan, this indicated the presence of
physiological races of the fungus. Singh and Reddy(1990) also reported that
because of numerous races of A. rabiei, it would be difficult to cultivars that
are resistant across all locations.
4.10.3 Chemical control
4.10.3.1 Food poisoning technique for the fungicides
From table(4-9) represents the effect of fungicides on the colony
diameter(mycelium growth), it seems that there are significant differences in
both mycelium growth and percentage of inhibition among treatments
Mancozeb, Rovral, and the control and these three treatments, significantly
varied with the other treatments. The fungicides, Domark, Benlate, Topas,
Dividend, Rubigan, and Raxil, inhibited the growth at the rate of 100
%(figure 4-8). Mancozeb inhibited mycelium growth at the rate of 47.25
while Rovral have the lowest inhibition, which was 37.20 %. In this aspect,
Abdou et al.,(1991a) revealed that invitro studies, Benlate inhibited A. rabiei
growth at 10 PPM. Results of Inam ul Haq et al.,(1995), seem to confirm the
present finding, However, they found that A. rabiei was most sensitive to
86
Chapter 3. Results and Discussions
Penconazole(as Topas C-50) and it is completely inhibited the growth invitro
at 50µg/ml.
Table(4-9): The effect of some fungicides on the mycelium growth of
A. rabiei in vitro.
Fungicides
Colony diameter(mm)
Domark
Benlate
Mancozeb
Topas
Rovral
Dividend
Rubigan
Raxil
Control
0.00*
0.00
11.50
0.00
13.66
0.00
0.00
0.00
21.83
d**
d
c
d
b
d
d
d
a
% of inhibition
100.00
100.00
47.25
100.00
37.20
100.00
100.00
100.00
0.00
a
a
b
a
c
a
a
a
d
* Each value represents means of four replicates
** Values within a column followed by a common letter are not significantly different
at P=0.01, according to Duncan,s multiple range test.
A
B
C
D
E
F
G
Figure (4-7): Response of seven chickpea cultivars inoculated with
Ascochyta rabiei in greenhouse: A: Dijla B: Shami C: Ghab-3 D: IPA-510 E: Harir
F: Ghab-2 G: Marakishi
87
Chapter 3. Results and Discussions
Rubigan 4%
Benlate 50%
Control
Domark 10%
Rovral 50%
Dividend 3%
Mancozeb 80%
Topas 10 %
Raxil DS 2%
Figure (4-8): Effects of some fungicides on the mycelium growth of A.
rabiei grown on CSDMA medium.
4.10.3.2 Seed treatment
4.10.3.2.1 Invitro studies
The results of seed treatment in vitro, showed that the fungicides Raxil,
Benlate, Mancozeb, and Dividend, eradicated the disease completely from
infected seeds(table 4-10). Rovral showed weak protection of infected seeds,
and it resulted in 13.33 % of A. rabiei colony formation, but has significant
differences with the control and with the other fungicides. The fungicides,
Raxil, Dividend, Benlate, Mancozeb, and Rovral did not differ significantly
with one another in percentage of seed Germination, but have significant
differences with the control. Rovral did not differ significantly with the
untreated control and caused lowering of germination because the fungus
88
Chapter 3. Results and Discussions
caused seed decay of some infected seeds or seedlings which prevented the
germination(figure 4-9). The results of Kaiser and Hannan(1988), seem to
confirm our results that in laboratory tests, the incidence of seed-borne A.
rabiei reduced from 45% in untreated to 0% in treated seeds by using Benlate
and thiobendazole(TBZ).
Table (4-10): The effect of seed dressing fungicides on appearance of A.
rabiei colonies and germination of chickpea seeds invitro.
Fungicides
% of colony formation
% of seed germination
Raxil
0.00* c**
100.00 a
Benlate
0.00
c
96.66
a
Mancozeb
0.00
c
93.33
a
Dividend
0.00
c
93.33
a
Rovral
13.33 b
83.33
ab
Control
93.33 a
66.66
b
* Each value represents means of four replicates
** Values within a column followed by a common letter are not significantly different
at P=0.01, according to Duncan,s multiple range test.
89
Chapter 3. Results and Discussions
Healthy
Infected
seedling
seedling
Figure(4-9): Effect of some seed dressing fungicides on the seedling
infection, Ascochyta colony formation, and germination of
chickpea seeds in vitro.
90
Chapter 3. Results and Discussions
4.10.3.2.2 In vivo studies
The results of seed treatment in greenhouse revealed that Raxil,
Benlate, and Dividend eradicated the pathogen A. rabiei from seeds
completely and has no significant differences with the healthy seed treatment.
Rovral and Mancozeb did not protect the seedlings from infection and they
gave seedling infection 8.75 and 18.13%, respectivly(table 4-11). Mancozeb
differed significantly with the healthy seeds treatment in percentage of
seedling infection but did not differ significantly with untreated
control(infected seeds) and with Rovral. In this aspect Kaiser and
Hannan(1988) confirmed that A. rabiei was transmitted to seedlings from
infected seeds planted in sterile soil under greenhouse conditions.
Maden(1983) also showed that transmission of A. rabiei to the arial parts of
chickpea seedlings from naturally infected seeds was 25% in greenhouse and
12.2% in the field. Statements that of AL-Taee(1997) seems to confirm our
findings that Benlate eradicated the disease from seeds completely. The
percentage of Germination was not affected by the seed treatment. There were
no significant differences between untreated control and healthy seed
treatment, and also with Raxil, Benlate, and Rovral. The lowest percentage of
Germination observed with the Mancozeb which have no significant
differences with Rovral, and this is probably due to that both Mancozeb and
Rovral did not protected the seeds completely. From Table(4-11) we observe
that germination speed was not affected with seed treatments, therefore there
were no significant differences among Benlate, Rovral, Dividend, and healthy
seeds in one side and untreated control in the other side, but both Raxil and
Mancozeb which have high germination speeds, differed significantly with
the other treatments with no differences with each other.
91
Chapter 3. Results and Discussions
Table(4-11): The effect of some seed dressing fungicides on the control of
A. rabiei, germination and germination speed of chickpea seeds invivo.
treatment
% of seedling
infection
Raxil
0.00* c**
96.25
ab
3.28 a
Mancozeb
18.13 ab
85.00
c
3.43 a
Benlate
0.00
c
96.25
ab
2.26 bc
Rovral
8.75
b
91.25
bc
2.00 c
Dividend
0.00
c
98.75
a
2.69 b
Cntrol(untreated
infected seeds)
24.55 a
92.50
ab
2.25 bc
Control
(healthy seeds)
0.00
95.00
ab
1.95 c
c
% of seed
germination
Germination
speed
* Each value represents means of four replicates
** Values within a column followed by a common letter are not significantly different
at P=0.05, according to Duncan,s multiple range test.
4.10.3.3 Foliar Application
4.10.3.3.1 In the greenhouse
The results of chemical foliar application in greenhouse revealed that
there were significant differences among treatments in degree of
infection(table 4-12and figure 4-10). The best control of the disease was
obtained with Raxil which reduced the degree of infection on the rate of
92
Chapter 3. Results and Discussions
53.70% compared to untreated control, followed by Topas which reduced the
degree of infection on the rate of 52.17%. Moreover, the degree of infection
of these two treatments did not reach to the susceptible level which was >2.5
in a 0-5 scale, but other treatments, Benlate, Rubigan, and Domark, inspite of
their significant differences with the untreated control, did not prevent the
plants to reach the susceptible point. The table also shows that the lowest
percentage of stem infection was with Topas which was 2.93%, followed by
Raxil and Domark which have percentages of stem infection of 3.66 and
17.2%, respectively, and these three treatments did not differ with one
another, but differed significantly with the untreated control.
The results showed that Topas came first in plant height followed by
Raxil with no significant differences with each other, but differed
significantly with the other treatments. The present study in table(4-12) also
shows that both fungicides, Raxil and Topas, came first in dry weight and
these two treatments differed significantly with the other treatments.
We concluded that in addition of that all fungicides used in this
experiment completely inhibited the growth of the pathogen in food poisoning
technique(invitro studies table 4-9), but in this study, none of these fungicides
completely eradicated the pathogen from the plants. In agreement with our
results, Islam et al.,(1999) argued that Topas C-50 suppressed the rate of gram
blight disease development compared to untreated control, and they
confirmed that none of the fungicides used in the trial applied once or twice,
completely inhibited the symptoms development.
93
Chapter 3. Results and Discussions
Table(4-12): The effect of some fungicides used as post infection spray to
control the Ascochyta blight of chickpea in greenhouse.
Treatment
Degree % Reduction of
% of stem
of
degree of
infection
infection
infection.
Plant
height(cm)
Dry
weight(g)
Raxil
2.13*e
53.70
3.66 d
22.87 a
0.89 a
Topas
2.20 de
52.17
2.93 d
23.60 a
0.85 a
Benlate
3.60 b
21.74
55.66 b
17.47 c
0.63 b
Rubigan
3.00 c
34.78
39.66 c
19.60 bc
0.70 b
Domark
2.60 cd
43.48
17.20 d
20.13 b
0.72 b
Control
4.60 a
0.00
74.00 a
18.47 bc
0.62 b
*Each value represents means of three replicates.
** Values within a column followed by a common letter are not significantly different
at P=0.05, according to Duncan,s multiple range test.
A
B
D
C
E
F
Figure(4-10): Response of chickpea plants infected by A. rabiei to some
fungicides used as a foliar application in greenhouse
A: untreated control, B: Domark 10% EC, C: Rubigan 4% EC, D: Benlate
50% WP, E: Topas 10% EC, F: Raxil 2DS.
94
Chapter 3. Results and Discussions
4.10.3.3.2 In the field
The results of foliar application in the field are obtained by using five
different fungicides, which were Raxil, Topas, Domark, Rubigan, and
Benlate. It revealed the fact that there were no effects of any fungicides in
controlling the disease with the susceptible cultivar Shami. Though, there
were no significant differences among any of treatments and with the
control(table 4-13), and this is probably because of the intermittent rain for
long period which continued from 18 of March until 7 of Aprril, therefore, the
water splashes created many secondary infections during this period and
resulted of no fungicides effects. We concluded that three sprays were
insufficient under rainy conditions with the susceptible cultivars. The present
result are in agreement with previous reports of Reddy and Singh(1990c) that
more than six fungicides applications per season are needed to control the
disease. Other studies that support our results reported by Reddy et al.,(1992)
who found that Most of fungicides were less useful for application during
rains. It should be mention that the plants in all treatments were devastated by
the disease and not reached to the yield stage therefore, only degree of
infection was measured and other variables that mentioned in the methods
were not measured.
95
Chapter 3. Results and Discussions
Table (4-13): The effect of post infection application of fungicides in the
field on the Ascochyta blight development during 2002 season.
Treatment
Degree of infection
Raxil
4.59
a
Topas
4.40
a
Benlate
4.44
a
Rubigan
4.51
a
Domark
4.64
a
Control
4.53
a
* Each value represents means of three replicates.
** Values within a column followed by a common letter are not significantly different at P=0.05,
according to Duncan,s multiple range test.
96
MSc. Thesis – Qasim Marzani
References
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