Botryodiplodia sp. - Crops for the Future

Control of Post Harvest Disease 

(Botryodiplodia sp.) of Rambutan and Annona 

Species by Using a Bio-Control Agent 

(Trichoderma sp.) 

Robert Kunz 

June 2007

List of Contents 

List of Contents .......................................................................................................................... 1 

List of Figures ............................................................................................................................ 2 

List of Tables.............................................................................................................................. 3 

List of Acronyms........................................................................................................................ 3 

1 Summary ................................................................................................................................. 4 

2 Introduction ............................................................................................................................. 5 

3 Sri Lanka ................................................................................................................................. 6 

3.1 Geography ........................................................................................................................ 6 

3.2 Climate ............................................................................................................................. 8 

3.3 Socio-economics .............................................................................................................. 8 

4 The host institutes.................................................................................................................... 9 

4.1 International Centre for Underutilised Crops (ICUC)...................................................... 9 

4.2 Industrial Technology Institute (ITI)................................................................................ 9 

5 Reason for research project ................................................................................................... 10 

6 The plants .............................................................................................................................. 11 

6.1 Rambutan (Nephelium lappaceum)................................................................................ 11 

6.1.1 Description of the plant/fruit ................................................................................... 11 

6.1.2 Main diseases .......................................................................................................... 13 

6.1.3 Economics in Sri Lanka .......................................................................................... 13 

6.2 Annona (Annona sp.)...................................................................................................... 13 

6.2.1 Description of the plant/fruit ................................................................................... 14 

6.2.1.1 Annona cherimola ................................................................................................ 14 

6.2.1.2 Annona muricata .................................................................................................. 16 

6.2.1.3 Annona squamosa................................................................................................. 17 

6.2.1.4 Annona reticulata................................................................................................. 18 

6.2.2 Main diseases .......................................................................................................... 19 

6.2.3 Economics in Sri Lanka .......................................................................................... 19 

7 The disease (Botryodiplodia sp.)........................................................................................... 19 

7.1 Taxonomy....................................................................................................................... 19 

7.2 Disease pattern ............................................................................................................... 21 

8 Bio-control agent (Trichoderma sp.)..................................................................................... 21 

8.1 Taxonomy....................................................................................................................... 21 

8.2 The role of mycoparasitism............................................................................................ 22 

9 Experiments........................................................................................................................... 23 

9.1 Experimental procedure ................................................................................................. 23 

9.1.1 Laboratory set-up ........................................................................................................ 23 

9.1.2 Soil sampling........................................................................................................... 25 

9.1.3 Isolation of the bio-control agent ............................................................................ 26 

9.1.4 Isolation of the pathogen ......................................................................................... 28 

9.1.5 Bio-Assays............................................................................................................... 29 

9.1.6 Cross-inoculation..................................................................................................... 30 

9.1.7 Sporulation experiment ........................................................................................... 32 

9.2 Results ............................................................................................................................ 32 

10 Conclusion........................................................................................................................... 37 

11 Acknowledgement............................................................................................................... 37 

12 References ........................................................................................................................... 38 

13 Annexes............................................................................................................................... 41 

1

List of Figures 

Figure 1: The Sri Lankan Flag ..................................................................................... .... 6 

Figure 2: The Sri Lankan coat of arms ........................................................................ ..... 6 

Figure 3: Map of Sri Lanka ......................................................................................... ..... 7 

Figure 4: Different shapes of the rambutan fruit: 1) oval, 2) ovoid and 3) 

Ellipsoid......................................................................................................... .. 12 

Figure 5: 

The most important rambutan varieties in Sri Lanka: `Malwana Special´, 

`Malaysian Yellow´ and `Malaysian Red’ .................................................... .. 12 

Figure 6: Annona cherimola ......................................................................................... .. 15 

Figure 7: Annona muricata tree .................................................................................... .. 16 

Figure 8: Annona muricata ........................................................................................... .. 16 

Figure 9: Annona squamosa ......................................................................................... .. 18 

Figure 10: A red skinned Annona squamosa fruit ......................................................... . . 18 

Figure 11: Annona reticulate .......................................................................................... .. 18 

Figure 12: Mature and immature spores of Botryodiplodia sp. ...................................... .. 20 

Figure 13: 

Petri-dish with Botryodiplodia sp. after seven days (left) and after 

four Weeks (right) ....................................................................................... .. 20 

Figure 14: A. muricata fruit infected with diplodia rot................................................... .. 21 

Figure 15: Phialides of Trichoderma sp.......................................................................... .. 22 

Figure 16: Petri-dish with a six day old Trichoderma sp. Colony.................................. .. 22 

Figure 17: Trichoderma harzianum parasitizes Pythium ultimum.................................. .. 22 

Figure 18: Autoclave at ITI............................................................................................. .. 24 

Figure 19: Laminar of flow at ITI................................................................................... .. 24 

Figure 20: Incubator at ITI.............................................................................................. .. 25 

Figure 21: Soil sampling in a rambutan plantation......................................................... .. 26 

Figure 22: Seven days old plate from a soil experiment................................................. .. 27 

Figure 23: Isolation of the pathogen on rambutan (five days old plate .......................... .. 28 

Figure 24: Preparation of a Petri-dish for a bio-assay .................................................... .. 29 

Figure 25: BC-agent with a good (right) and with a bad (leftight) effectiveness ........... .. 30 

Figure 26: Experimental series of a bio-assay after two (left) and six (right) days........ .. 30 

Figure 27: Humidity chamber of the rambutan cross-inoculation experiment ............... .. 31 

Figure 28: Infestation key for the cross-inoculation experiment with sugar apple......... .. 31 

Figure 29: Experimental series of a cross-inoculation on A. squamosa 

(day 2 to day 6).............................................................................................. .. 32 

Figure 30: The twelve Trichoderma sp. found in the soil samples................................. .. 33 

Figure 31: 

The four Botryodiplodia sp. isolates which were used for all 

Experiments................................................................................................... .. 33 

Figure 32: Results of the cross-inoculation on rambutan ............................................... .. 34 

Figure 33: Results of the cross-inoculation on Annona muricata................................... .. 35 

Figure 34: Results of the cross-inoculation on Annona squamosa................................. .. 35 

Figure 35: Organisation structure of the ITI................................................................... .. 41 

Figure 36: List of the bio-control agents isolated ........................................................... .. 42 

2

List of Tables 

Table 1: An Overview of the soil samples which were taken……………………………….. 26 

Table 2: An overview of the isolated Trichoderma sp………………………………………. 33 

Table 3: An overview of the isolated pathogens…………………………………………….. 33 

Table 4: Results of the effectiveness of the isolated Trichoderma sp……………………….. 34 

Table 5: Results of the sporulation experiment……………………………………………… 36 

List of Acronyms 

BC-agent - Bio-control agent 

BPS - Berufspraktisches Projektsemester 

CGIAR - Consultative Group on International Agricultural Research 

CISIR - Ceylon Institute of Scientific and Industrial Research 

DOASL - Department of Agriculture, Sri Lanka 

GTZ - Deutsche Gesellschaft für Technische Zusammenarbeit 

HIV - Human immunodeficiency virus 

ICUC - The International Centre for Underutilised Crops 

IPGRI - International Plant Genetic Resources Institute 

ITI - Industrial Technology Institute 

IWMI - International Water Management Institute 

PDA - Potato Dextrose Agar 

UK - United Kingdom 

3

1 Summary 

Experiments were undertaken by the Industrial Technology Institute (ITI) in cooperation with 

the International Centre for Underutilised Crops (ICUC) to prove the availability of 

Trichoderma sp. in Sri Lanka and their effectiveness as Bio-control agents (BC-agents) 

against Botryodiplodia sp. 

The Trichoderma sp. used in the experiments were isolated from soil samples taken from 

several locations in Sri Lanka and the pathogens were isolated from diseased fruits of Annona 

squamosa, Annona muricata and rambutan (Nephelium lappaceum). 

Bio-assays were undertaken to prove the effectiveness of the BC-agents. In addition crossinoculation 

experiments were done to see whether the pathogens of various fruits infect each 

other. Furthermore a sporulation experiment was attempted to check if a reduction of the 

sporulation time of Botryodiplodia sp. under special treatments (cold shock and daylight) is 

possible. 

Following results of the experiments are shown: 

Isolates: 

Twelve isolates of Trichoderma were obtained from six different soil-sources and eight 

isolates of the pathogen from diseased fruits. One pathogen from each fruit was taken for 

further experiments. 

Bio-assay: 

The bio-assay experiment showed that three of the Trichoderma sp. isolates have a high, four 

have moderate and three have low Bio-control activity while three isolates observed to be non 

effective. Two of the Trichoderma strains with a high BC-activity were isolated from 

rambutan orchards in Warakapola and the third was found in a soil sample, taken from the 

surrounding area of an Annona muricata tree in Wijayapura. 

None of the isolates taken from Annona squamosa sources showed high BC-activity, while 

one was moderately effective against the pathogens. 

Cross-inoculation experiment: 

The pathogens which were cross-inoculated to the rambutan fruits showed an equal severity 

after the third day. On the first two days the severity that was caused by the pathogen isolated 

from Annona muricata was a little higher. 

4

The cross-inoculation experiment with Annona muricata fruits did not lead to satisfactory 

results. The reason being that the severity of infection in the two controls were the same or 

higher to the severity caused by the pathogens. A statistically significant higher severity 

between pathogen taken from Annona muricata and the one taken from Annona squamosa 

compared to the others can be seen at the fifth day. 

The variability in the cross-inoculation experiment with Annona squamosa fruits was high; 

nevertheless we can conclude that the pathogens of the various fruits do infect the others. 

Sporulation experiment: 

The treatment with a cold shock resulted in the longest sporulation times of 26 days with 

respect to all pathogens which were observed; the pathogen isolated from a red skinned 

Annona squamosa which did not sporulate during the experimental period. The pathogens 

which were isolated from rambutan and Annona squamosa sporulated fastest (15 days) under 

the daylight treatment. The pathogens isolated from the red skinned Annona squamosa and 

from Annona muricata sporulated fastest (24 days) in the incubator without any treatment. 

The two collaborative Institutes are described in this report as well as the country. Characteristics 

of both fruits used for the experiments and other important Annona species (A. cherimola and A. 

reticulata) as well as the two fungi are also described. 

2 Introduction 

The background for the research presented here was the “Berufspraktisches Projeksemester” 

(BPS) programme which has to be done as a practical part of the author’s studies towards a 

Bachelor of Science degree in Horticulture-Management. 

The work was done in two institutes in Sri Lanka: The International Centre for Underutilised 

Crops (ICUC) which has announced the internship and the Industrial Technology Institute 

(ITI) which was the main working place for the experimental trails. 

The Intern was involved in a project of ITI and conducted several experiments which are 

described on the following pages. 

The main task of the intern was to prove antagonistic effects of fungus (Trichoderma sp.) 

against a fungal pathogen (Botryodiplodia sp.). 

5

3 Sri Lanka 

The Democratic Socialist Republic of Sri Lanka is an insular state in the Indian Ocean and 

was earlier known under the name Ceylon. Sri Lanka is a member state of the Commonwealth 

with the seat of the government is Sri Jayewardenepura and the capital as well as the biggest 

city is Colombo. 

The following Figures (1 and 2) are showing the flag and the coat of arms of Sri Lanka. 

Figure 1: The Sri Lankan flag (MY WORLD GUIDE, 2007) 

Figure 2: The Sri Lankan coat of arms (MY WORLD GUIDE, 2007) 

3.1 Geography 

Sri Lanka is a tropical island lying close to the southern tip of India and near the Equator 

between 6° and 10° northern latitude and between 79° and 82° eastern longitude. It is parted 

from India through the Palk Strait and the Gulf of Mannar. The coral islands of the Adam’s 

Bridge represent an incoherent connection of the north-western Sri Lanka and the Indian state 

Tamil Nadu. 

6

The longest destination from the north to the south is about 440 km and the widest part of the 

island measures around 220 km. The total expanse of the country is 65,610 km² and the 

highest elevation of the central highlands is the Pidurutalagala with 2524 m above sea level. 

Sri Lanka is commonly divided in three different landscapes: the central highlands, the 

lowland plains and the coastal areas (Figure 3). 

Figure 3: Map of Sri Lanka (THE UNIVERSITY OF TEXAS, 2000) 

7

3.2 Climate 

The climate of Sri Lanka is tropical with different precipitation rates which accrue due to the 

effects of the monsoons. While the southwest of the country is humid with two precipitation 

maxima in May and October, the southeast monsoon brings only little rainfall on the northeast 

and east coast because this area is in the lee of the central mountains. In these areas most 

precipitation falls due to the northeast monsoon in November and December. 

The mean annual temperature is 22.2 °C in Kandy, 27.8 °C in Colombo and 33 °C in 

Tricomalee. 

The natural vegetation of Sri Lanka varies according to climatic zone and elevation. Dense 

evergreen rain forests are found in the south-western lowlands. In the central highlands, 

evergreen mountain forests are interspersed with grasslands. The drier evergreen forests in the 

north and east contain trees such as ebony and satinwood. Thorn forests and drought-resistant 

shrubs prevail in the driest areas. Along the coast, mangrove forests border lagoons and river 

estuaries. 

3.3 Socio-economics 

The population of Sri Lanka is about 20 million (AUSWAERTIGES AMT, 2007) and ethnically 

heterogeneous. 74.6 % of the population are Singhalese, 18.1 % are Tamil, 7 % are Arabians 

and 0.3 % are others. 

The Tamils were deployed from the British during the colonial time mainly as workers at tea 

plantations or as workers in the administration which led to a favouritism of the Tamil 

population compared to the Singhalese population. This caused, after the independency of the 

country in 1948, great antipathies between the two population groups, whereupon the Tamil 

population of the north and the east tried first peacefully then with force of arms to create 

their own state. This was the beginning of the civil war which still is a continuous armed 

conflict. 

Sri Lanka is also a country of religious diversity. Most of the population are Buddhists (69.3 

%) which are mainly Singhalese people. 15.5 % are Hindus where most of them are Tamil 

people followed by Christians (Singhalese and Tamils) with 7.6 % of the population and last 

Muslims with 7.5 %. 

According to the AUSWAERTIGE AMT (2007) the economy of Sri Lanka has risen in the year 

2005 about a value of 5.3 % and for the year 2006 again a high economic growth was 

expected. The economical development of the country shows high regional differences. The 

8

economic centre is the region around Colombo which achieves nearly half of the total 

economic performance. 

The economic change of structure shows that the agricultural sector (tea, rubber and coconut) 

was dominating once, but at present only makes up 18 % of the economical overall 

performance. 26 % are contributing by industry, especially the textile sector. The service 

sector makes up 55 % of the economical performance especially the tourist sector. 

In the agricultural sector the tea industry has a special relevance. The exports of tea yield 

about 20 % of the foreign exchange proceeds. The tourist sector has lost on relevance due to 

the Tsunami in 2004 and due to the upcoming ethnic conflict. 

4 The host institutes 

4.1 International Centre for Underutilised Crops (ICUC) 

The International Centre for Underutilised Crops (ICUC) is a global research, development 

and training organisation which was established in 1992. It is a Partner Organisation to the 

Consultative Group on International Agricultural Research (CGIAR). 

Since 2005 ICUC is located in the complex of buildings of the International Water 

Management Institute (IWMI) in Sri Lanka (Colombo) and is working there independently. 

To describe the activities of ICUC the term “underutilised crops” has to be defined first: 

According to ICUC (2006) underutilised crops “are plant species which are used traditionally 

for their food, fibre, fodder, oil or medicinal properties. They have an under-exploited 

potential to contribute to food security, nutrition, health, income generation and environmental 

services.” 

The mission of the institute is “to promote the use of underutilised crops for the benefit of humankind 

and the environment to reduce poverty and suffering through the improvement and 

promotion of underutilised crops.” 

ICUC works in collaboration with many national and international partners and acts as a 

knowledge hub for tropical, sub-tropical and temperate plant development. 

The Industrial Technology Institute (ITI) is one of the partners ICUC is working with. 

4.2 Industrial Technology Institute (ITI) 

The Industrial Technology Institute (ITI) is a Statutory Board, which came into existence as 

successor to the Ceylon Institute of Scientific and Industrial Research (CISIR) on 1998. The 

ITI functions within the purview of the Ministry of Science and Technology. ITI provides 

9

valuable consultations in industrial, scientific and technological areas and offers the major 

analytic services which are necessary for the public and private sector industry. Among other 

things food- and product assays, soil- and water analyses and functional tests are carried out at 

ITI (ITI, 2001). 

In 1991 the research and development policies and strategies were reformulated as the 

demands of industry and other private sectors have changed. This new policy underlined the 

need to be market oriented and demand driven with respect to its research and development 

and service function with a view to being self-financing. 

Today the ITI maintains its premier role as an interdisciplinary research institute in Sri Lanka 

dedicated to the promotion of industrial development through research, consultancy and other 

technical services and stands committed to serve the nation to meet the challenges of the 

future (ITI, 2007). 

The ITI has about 355 staff and is structured in six divisions, which consist of specialized 

groups (Annexes, Figure 34). The author main working place was located in the Post Harvest 

Technology Group of the Agro and Food Technology Division. The group consists out of 

eight people. 

5 Reason for research project 

Recent studies have shown the importance of products of underutilised fruits, such as jams, 

juices and candied fruits with regard to nutrition, income generation and poverty reduction of 

small scale entrepreneurs in developing countries (AZAM-ALI, 2003). Underutilised tropical 

fruits such as rambutan (Nephelium lappaceum) and annona (Annona sp.) provide important 

contributions to small-holder livelihoods. However, heavy post-harvest losses significantly 

reduce the full potential for income generation. 

The Post Harvest Technology Group of ITI is working on a project which develops biological 

non chemical methods for control of post harvest diseases of tropical fruits such as mango, 

banana and papaya as well as working together with ICUC on promising underutilised species 

such as rambutan and annona. The project presented here was part of this research program. 

The stem-end rot is caused by the fungus Botryodiplodia sp. and is one of the major post 

harvest diseases on many fruits (GUPTA et al., 1999, PLOETZ, 2003 and ALAM et al., 2001). 

Among others it counts to the most important pre- and post harvest diseases on rambutan 

(SIVAKUMAR et al., 1997) and annona species (DE Q. PINTO, 2005). The use of the mycoparasitic 

fungus Trichoderma sp. as a bio-control agent represents an alternative plant protection 

10

measure to chemical treatments. The effectiveness of Trichoderma sp. against certain fungi 

has been proven in a number of experiments (SIVAKUMAR et al., 2000, BARBOSA et al., 2001 

and WANTOCH-REKOWSKI, 2004). 

The following three points were the main task of the study which is described in this report: 

• To establish whether bio-control agents are widely available in Sri Lanka. 

• To prove the efficacy of the bio-control agents against the pathogen. 

• To prove whether diseased fruits of various species infect each other. 

6 The plants 

6.1 Rambutan (Nephelium lappaceum) 

Rambutan belongs to the family of Sapindaceae. Its common botanical name is Nephelium 

lappaceum, but there are two other synonym names: Euphoria nephelium and Dimocarpus 

crinita (MORTON, 1987a). The family Sapindaceae includes many fruit species but only 

rambutan, litchi (Litchi chinensis) and longan (Euphoria longana) have commercial importance 

(REHM and ESPIG, 1984). 

The origin of the fruit is Malaysia and it is mostly cultivated in Southeast Asia. A few 

rambutan trees are also grown in the costal lowlands of Colombia, Ecuador, Honduras, Costa 

Rica, Trinidad and Cuba. Some trees are also cultivated in Australia. (LAKSMI et al., 1987). 

Rambutan flourishes best in humid tropical regions with well distributed rainfall (2500-3000 

mm) and in deep soils with high organic matter. The plant grows from sea level to 500 m 

height and needs temperatures over 10 °C. The dry season should not last more than three 

months (REHM and ESPIG, 1984). According to MORTON (1987a) the Oriental Mindora region 

of the Philippines with an average temperature of about 27.3 °C, a relative humidity of 82 % 

and about 165 rainy days is the ideal environment. 

The name rambutan comes from the Malayan word “rambut” and means hairy, which 

correlates with the appearance of the fruit. 

6.1.1 Description of the plant/fruit 

The height of the rambutan tree varies between the different varieties and the method of 

cultivation from 8 to 25 m (BAERTELS, 1990, FRANKE, 1994 and MORTON, 1987a). The 

evergreen tree has alternate pinnate 7 to 30 cm long leaves with 1 to 4 pairs of leaflets. The 

leaflets are elliptic to oblong, pale shining with a dark-green colour. 

11

Rambutan trees are either dioecious or monoecious with male or hermaphrodite flowers and 

need cross pollination for fruit set. The petal-less flowers are very inconspicuous because of 

their tiny size (2.5 to 5 mm) and their greenish colour. They are growing on long shafted and 

much branched, to 30 cm long panicles and have a sweet smelling fragrance. 

The fruit is maturing in 15 to 18 weeks after flowering and gets an ovoid, round or ellipsoid 

shape with a pinkish-red, bright- or deep-red, orange-red, maroon or dark-purple, yellowishred 

or yellowish colour (Figure 4). The size of the rambutan fruit varies from 3.4 to 8 cm 

(BAERTELS, 1990, FRANKE, 1994 and MORTON, 1987a). The fruit has a thin parchment like 

rind with tubercles from each of which extends a soft spine. These hairy spines are 0.5 to 2 

cm long and have a red, pinkish or greenish-yellow colour (Figure 5). 

Figure 4: Different shapes of the rambutan fruit: 1) oval, 2) ovoid and 3) ellipsoid (IPGRI, 2003) 

Figure 5: The most important rambutan varieties in Sri Lanka: `Malwana Special´, `Malaysian 

Yellow´ and `Malaysian Red 

The white or rose-tinted and translucent 0.4 to 0.8 cm thick flesh is very juicy and covers one 

ovoid to oblong almond-shaped dark seed which is 2.5 to 3.5 cm long and 1 to 1.5 cm wide. 

Rambutan fruits have an exotic aroma which results out of the interaction of fruity-sweet and 

fatty-green odours, with the possible contribution of civet-like sweetish, spicy and woody 

notes (ONG, ACREE and LAVIN, 1998). 

12

6.1.2 Main diseases 

The most important yield reducing factor in rambutan plantations are birds and flying foxes 

and some other non flying mammals that consume many of the fruits (MORTON, 1987a). 

According to MORTON (1987a) the major pests are leaf eating insects like the mealy bug 

(Pseudococcus lilacinus) and the giant bug (Tessaratoma longicorne). The Oriental fruit fly 

attacks very ripe fruits and counts, according to FRANKE (1994), to the most important pests. 

There are several pathogens that attack the fruit and cause rotting under warm and moist 

conditions, like Botryodiplodia sp. which was mentioned before. These fungi are important 

post harvest diseases. 

Oidium sp. Phomopsis sp. and other soil born fungi maintain damage on foliage and other 

parts of the tree. Fomes lignosus is causing the stem canker in the Philippines and can be fatal 

for rambutan trees if not controlled (MORTON, 1987a). 

6.1.3 Economics in Sri Lanka 

There is not much information available about the economics of rambutan in Sri Lanka. 

However, rambutan is grown on medium scale commercial cultivation in Sri Lanka and the 

fruit has a high local demand (DOASL, 2007 and BARRY, 2007). The only data which could 

be found about the cultivation of rambutan are within the years 1995 to 1999 and show 

significant growth of the cultivation of rambutan with a production of 7168 metric tons and 

total land extent of 896 ha in the year 1999 (DOASL, 2007). 

Rambutan is a crop best suited to the mid-country and low-country wet zone of Sri Lanka. 

The main harvest season is July/August and sometimes, depending on the weather conditions, 

a small off-season harvest is gathered during December to February. 

The crop has good export potential and is presently exported in very limited amounts to the 

Gulf Region, France, Germany and UK. The most important variety is `Malwana Special´ 

followed by `Malaysian Red´ and ’Malaysian Yellow´ and some other local cultivars. 

Presently the most important areas where rambutan is grown successfully are within the 

districts of Gampaha and Colombo (DOASL, 2007). 

6.2 Annona (Annona sp.) 

Annona belongs to the family Annonaceae which number of genera and species is still 

debated. Thereby varies the number of genera from 40 to 119 and the number of species from 

500 to over 2000 (DE Q. PINTO et al., 2005, BAERTELS, 1990 and MAHDEEM, 1994). 

13

However, the most important genus is Annona which has a multitude of species. Most of 

these species are originated to tropical America and only a few are native to tropical Africa. 

This report deals with four species which are selected by ICUC (2002) as relevant 

underutilised plants: Annona cherimola, A. muricata, A. squamosa and A. reticulata. Only A. 

muricata and A. squamosa were tested in the experiments. 

A. cherimola, A. muricata and A. squamosa have great commercial importance whereas A. 

reticulata is significant a local used crop. They all belong to the areas of tropical South 

America although they can be found in most tropical and some subtropical regions around the 

world. Especially A. cherimola which can be cultivated in cooler and drier climates is widely 

spread in subtropical areas. According to DE Q. PINTO et al. (2005) have A. muricata and A. 

squamosa the widest distribution. 

Annona trees are multipurpose trees. Not only the fruit is consumed widely but the tree is also 

a source for medical and industrial products. The most important processed products are juice, 

jam and ice-cream (ICUC, 2002 and PRASADA RAO, AZEEMODDIN and THIRUMALA RAO, 

1984). 

Seed oil can be won out of all species and contains a large number of chemical compounds, 

like flavonoids, alkaloids and acetogenins. Therefore the oil is used for treatments of 

medicinal conditions, for example skin diseases, intentional worms, inflammation of the eye 

and anti HIV and cancer effects. 

6.2.1 Description of the plant/fruit 

Annona trees are mostly shrubs or small trees with an erect or spreading tree crown and a 

grey-brownish, rough and corrugated bark. Most Annona species are deciduous trees, 

especially when cultivated in areas with a dry season and without irrigation. The size, shape, 

colour and taste of the fruits are very diverse between the single species. 

6.2.1.1 Annona cherimola 

According to MORTON (1987b) the fruit of Annona cherimola is the most appreciated fruit of 

the genus Annona. The common name is cherimoya but the name is often misapplied to the 

custard apple (Annona reticulate) and to atemoya, which is a hyprid of A. cherimola and A. 

squamosa. 

The origin of the fruit is the tropical highland of Peru, Ecuador, Columbia and Bolivia where 

it was cultivated as an ancient crop by the Incas. Between 1400 and 2000 m above sea level 

with a temperature between 17 °C and 20 °C are the regions where cherimoya naturally 

14

grows. Nowadays cherimoya is grown in most countries of the tropical highland and the 

subtropics, like Spain, Egypt and Italy. In 1880 the fruit was introduced to Sri Lanka (Ceylon) 

where it is cultivated at small scale (MORTON, 1987b). 

The cultivation of cherimoya has commercial importance in Chile, Bolivia, Spain, United 

States (Florida) and New Zealand (GEORGE and NISSEN, 1991). 

The deciduous cherimoya tree is small, erect and somewhat spreading and reaches a height of 

5 to 9 m. It has a shrub-like appearance, because of the in ground level frequently divided 

stem. 

The alternate leaves are 2 to 4 ranked and ovate-lanceolate to elliptical in shape with tiny and 

hairy petioles. The leaves are 10 to 25 cm long, 4 to 9 cm wide, slightly hairy on the upper 

surface and brownish velvety-tomentose on the underside. The flowers have a good fragrance 

and grow extra-axillary mostly solitary and opposite a leaf at the base of a branchlet. 

The flowers have a white-pinkish colour and are quite small. The 

shape of the fruit is normally conical, oval or somewhat heart 

shaped (Figure 6). But the form is very varied due to irregular 

pollination. The fruit is 10 to 15 cm long, 5 to 10 cm wide and 

has an average weight of 150 to 700 g. In most varieties the 

surface is covered with small conical protuberances over the 

carpel. The skin of the fruit is thin and has a greenish-yellow 

colour when fully ripe. The pulp is snow white, juicy and contains 

numerous hard, brown, 1.2 to 2 cm long and glossy seeds. The 

Figure 6: Annona 

cherimola fruit has a pleasing aroma and delicious, sub acid and fragrant 

flavour, like a mix of pineapple and banana (MORTON, 1987b and 

DE Q. PINTO et al., 2005). The male and female parts of the flower do not mature 

simultaneously. That is the reason of the inadequate natural pollination of cherimoya. To 

reach a high fruit set rate the farmers are forced to do hand pollination (MORTON, 1987b). 

15

6.2.1.2 Annona muricata 

Annona muricata is the species out of the genus Annona which produces the biggest flowers 

as well as the biggest fruits. The common English name is soursop and the Sri Lankan call the 

plant “katu anoda”. There is no clarity about the exact origin of the fruit. Most authors opine 

that soursop is native to Central America, the Antilles or the 

northern South America (DE Q. PINTO et al., 2005, MORTON, 

1987b and MAHDEEM, 1994). The soursop was one of the first 

fruits carried out from South America to the Old World 

tropics where it has become widely distributed, from 

southeaster China to Australia and the warm lowlands of 

eastern and western Africa. It is also found in Sri Lanka up to 

en elevation of 460 m above sea level (DE Q. PINTO et al., 

2005). 

The soursop tree has a height of 4 to 10 m and is described as 

a low branching and bushy but slender tree (Figure 7). The 

evergreen tree has alternated, smooth and glossy leaves with 

short petioles (3 to 7 mm long). The upper surface is dark 

green whereas lighter beneath. The leaves are oblong-ovate to 

cylindrical, 6 to 20 cm long and 3 to 7 cm wide. 

Figure 7: Annona muricata 

tree 

The plant has very big flowers compared to the other Annona species, being 3 to 5 cm long. 

The greenish-yellow flowers born single and emerge anywhere on the trunk, branches or 

twigs. 


muricata 

The fruits of Annona muricata are oval or heart-shaped, 

sometimes irregular due to bad pollination (Figure 8). The size of 

the fruit is very variable in a range from 10 to 30 cm in length and 

up to 20 cm in width with a weight of 0.5 to 10 kg. The skin of the 

fruit has many short, fleshy and pointed protuberances and is 

popularly regarded as “spiny”. The colour is dark green when 

unripe and turns into a slighter yellowish-green when ripe. Its 

inner surface is cream-coloured and granular and separates easily 

from the mass of snow-white, cotton-fibrous, juicy segments 

surrounding the central, soft-pithy core. 

16

MORTON (1987b) describes the aroma of the fruit as follows, the pulp is somewhat pineapple 

like, but its musky, sub acid to acid flavour is unique. 

The fruits have a large number of oval, smooth, hard and black seeds, up to 170 in large fruits. 

6.2.1.3 Annona squamosa 

Sugar apple or sweetsop are common English names for Annona squamosa. The sugar apple 

has the Sri Lankan name “weli anoda” and is the most widely grown of all Annona species. 

The original home of the fruit is not really known. According to MAHDEEM (1994) Annona 

squamosa seems to be native in south eastern Mexico. MORTON (1987b) is of the opinion that 

the origin is unknown. DE Q. PINTO et al. (2005) explains that the plant is native to circum 

Caribbean or the northern South America lowlands. Today the tree can be found in nearly all 

tropical countries. 

Annona squamosa requires tropical or near tropical climates and has a high drought tolerance. 

It grows best in dry areas. In Sri Lanka the sugar apple flourishes in the wet as well as in the 

dry zones from sea level to 1100 m elevation (MORTON, 1987b). 

The deciduous tree reaches a height of 3 to 6 m with an open crown of lateral branches. The 

alternated leaves are lanceolate or oblong, blunt tipped, 5 to 17 cm long and 2 to 7 cm wide, 

with an obtuse apex. The colour is brilliant-green on the upper site and bluish-green with a 

bloom below. 

The flowers of the sugar apple trees are similar in size and form to the ones of the cherimoya 

tree. 

The fruit is globose, heart-shaped or conical in form with a diameter of 5 to 10 cm and a 

weight of 120 to 330 g (Figure 9). According to MORTON (1987b) the surface of the fruit, 

which is covered with tuberculates and a powdery bloom resembles a “hand-grenade.” The 

tuberculates are separating when the fruit is ripe so that the fruit falls to pieces. 

The creamy white and custard like pulp has a delightfully fragrant and a pleasant sweet-sour 

flavour. The fruit contains 35 to 45 seeds which are oblong-cylindrical, dark brown and about 

1.5 to 2 cm long. 

Pollination and fruit set problems are similar to those of other annonas. 

Annona squamosa has a few cultivars including some varieties with a red coloured skin, like 

A. squamosa `Red´, A. squamosa `Red-speckled´ and A. squamosa `Crimson´ (Figure 10). 

17

Figure 9: Annona squamosa 

Figure 10: A red skinned variety 

of Annona squamosa 

6.2.1.4 Annona reticulata 

DE Q. PINTO et al. (2005) considers that Annona reticulata is the most vigorous of the annona 

species described in this report. MORTON (1987b) however, reports that both in tree and in 

fruit the species is generally rated as the mediocre or “ugly duckling” species among the 

prominent members of the genus Annona. 

The common English name of Annona reticulata is bullock’s-heart or custard apple. 

In the past many people thought that the origin of the custard apple is West India but today it 

is proven that the plant is native to Central or northern South America (MORTON, 1987b, DE 

Q. PINTO et al., 2005 and MAHDEEM, 1994). Today the tree is 

found like the others afore described, in many tropical countries 

around the world. 

Annona reticulata grows well in tropical climates with cool 

winter times and prefers a humid atmosphere. The tree is 

tolerant to cold temperatures. According to MORTON (1987b) a 

fully grown custard apple tree survives temperatures of 2.8 °C 

without serious harm. 


reticulata 

The deciduous Annona reticulata tree is small and tender with an open and irregular crown 

which can reach a height of 5 to 10 m. The alternate leaves are oblong or narrow-lanceolate, 

dark-green and glabrous with a length of 10 to 25 cm and a width of 2 to 7 cm. 

The flowers are similar in form to those of Annona squamosa and Annona cherimola. The 

only different is that they are generally grown in drooping clusters with 2 to 10 flowers. The 

0.1 to 1 kg heavy fruit is commonly heart-shaped but sometimes different like oval, lopsided 

or nearly round (Figure 11). They are 8 to 15 cm in diameter, coriaceous and have a reddishyellow 

surface colour with impressed lines above the carpel. 

18

The flesh is creamy-white with a juicy aromatic to hard flavour with a repulsive taste 

according to the cultivar. According to DE Q. PINTO et al. (2005) the custard apple is the least 

tasty fruit of the cultivated annona species. 

The seeds which are dark-brown or black in colour, oblong, smooth and less then 1.25 cm 

long are commonly more than 40 in number. 

6.2.2 Main diseases 

According to DE Q. PINTO et al. (2005) are Annona trees attacked by a large number of insect 

pests and numerous diseases, although many of them are not economically important. The 

most important pests are aphids, mealy- and scale-bugs and fruit-flies (FRANKE, 1994). 

According to DE Q. PINTO et al. (2005) the annona moth (Cerconota annonella), commonly 

known as the “fruit borer” is the most important of the insect pests attacking Annona species. 

The most important root diseases caused by fungi are damping-off (Rhizoctonia solani and 

Fusarium spp.) and black root rot (Phytophthora spp., Cylindrocladium clavatum and 

Sclerotium rolfsii) (DE Q. PINTO et al., 2005). The bacterial wilt is also an important basal and 

root rot which affects mainly seedlings in nurseries and grafted trees in the field (PLOETZ, 

2003). There are several diseases attacking the fruits of Annona species during pre and post 

harvest phases: anthracnose (Glomerella cingulata), black cancer (Phomopsis 

annonacearum), diplodia rot (Botryodiplodia theobromae), purple blotch (Phytophthora 

palmivora) and brown rot (Rhizopus stolonifer) (DE Q. PINTO et al., 2005 and PLOETZ, 2003). 

6.2.3 Economics in Sri Lanka 

There is only little information available about the economic situation of annona species in Sri 

Lanka. In the course of his stay in Sri Lanka the reports author could not find any annona 

plantations. It seems that Annona trees are mostly planted in home gardens. The same 

situation could be seen on the markets where Annona species, especially soursop and sugar 

apple, were available but not in high quantities. By means of some market sellers cherimoya 

is imported to Sri Lanka in small quantities. 

7 The disease (Botryodiplodia sp.) 

7.1 Taxonomy 

Botryodiplodia sp. belong to the class of Ascomycetes and live in a saprophytic way. They are 

moulds which normally need injured tissue to parasite the plant. 

19

Characteristically for this fungus is the generation of pycnidia in which the spores of the 

fungus are formed. Pycnidia develop on artificial media only rarely and after a long time. But 

if they are generated they can be seen with the naked eye as black balls of the size of a 

pinhead. 

The spores are elliptical and compared to other spores relatively big. Young, immature spores 

are colourless and unicellular whereas mature spores are brown coloured, distichous and 

thick-walled (Figure 12). Only the presence of the mature spores allows a proper 

identification of the fungus. 

The fast and constantly growing mycelium of the fungus is snow-white at first, turning its 

colour within three to four weeks black (Figure 13). This discolouring is not due to the 

generating of the spores. 

The most important species of the genus is Botryodiplodia theobromae. 

Figure 12: Mature and immature spores of Botryodiplodia sp. 

Figure 13: Petri-dishes with Botryodiplodia sp. after seven days (left) 

and after four weeks (right) 

20

7.2 Disease pattern 

Botryodiplodia sp. is a fungal disease attacking the 

fruits of Annona species especially in neglected 

orchards. Diseased fruits show symptoms of 

purplish to black spots or blotches confined to the 

surface of the fruit and eventually covered with 

white mycelia and black pycnidia (Figure 14). 

Diplodia rot is distinguished by its dark internal 

discolouration and the extensive corky rotting 

produces (DE Q. PINTO et al., 2005). The 

penetrated flesh eventually softens or hardens and 

cracks, depending on the presence of secondary 

microbes. 

Figure 14: A.muricata fruit infected with 

diplodia rot 

8 Bio-control agent (Trichoderma sp.) 

Trichoderma sp. are fungi that are present in nearly all soils and other diverse habitats such as 

decaying wood. According to HARMANN (2005) the fungi are the most prevalent fungi in soil. 

They are favoured by the presence of high levels of plant roots, which they colonize readily. 

In addition to that Trichoderma spp. are able to attack, parasitize and otherwise gain nutrition 

from other fungi. These abilities turn the fungi into a reputable bio-control agent. 

8.1 Taxonomy 

The sexual life-form of the fungi of the genus Trichoderma is extensively unknown. So the 

classification of the fungus to the class of Deuteromycetes results from the asexual life-form. 

Reproduction of the ubiquitous genus is by conidia which are produced from highly branched 

conidiophores. These are arranged in irregular verticils, with the sub-terminal phialides borne 

more or less at right angles to the stipule. The shape of the phialides is typical for 

Trichoderma sp. and is the crucial factor for the identification of the fungi (PITT and 

HOCKING, 1985) (Figure 15). 

21

Figure 15: Phialides of Trichoderma sp. 

Figure 16: Petri-dish with a six day 

old Trichoderma sp. colony 

Trichoderma sp. colonies spread rapidly and have a loose textured mycelium which 

characteristically develops irregularly, with tufts or isolated patches. The mycelium has a 

white transparent colour. The small conidia are in general green coloured and smooth and 

develop after a short time of incubation. That is the reason why the mycelium is turning into 

green after a few days (Figure 16). 

8.2 The role of mycoparasitism 

As aforementioned Trichoderma spp. are already used as 

bio-control agents against phytopathogenic fungi and 

bacteria. Pythium, Phytophthora, Rhizoctonia, Fusarium 

and Verticillium are fungi against which Trichoderma sp. 

have already made an impact (KORTING, 2001). The 

reasons for this use are certain antagonistic attributes 

which most fungi out of the genus Trichoderma own. The 

most important mechanisms are, according to WANTOCH- 

REKOWSKI (2004), mycoparasitism, antibiosis and competition. 

Trichoderma sp. are able to enter into the mycelium 

of other fungi. Therefore the fungi wind around the 

host’s mycelium and build hyphae which break into the 

host’s mycelium (Figure 17). In this way the antagonists 

are able to “suck” the hosts. 

Figure 17: Trichoderma 

harisianum parasitizes Pythium 

ultimum from (BENHAMOU and 

CHET, 1997 and WANTOCH- 

REKOWSKI, 2004) 

22

The fungi are producing enzymes such as cellulases and chitinases which are able to destroy 

the cell walls of other fungi or inhibit the germination of other spores. Some Trichoderma 

strains compose antibiotics which can control bacterial phytopathogenics). These mechanisms 

are called antibiosis. 

According to WILKE (2001) Trichoderma provides the same compounds as plant fortifier 

because it produces hormones and enzymes which are supporting the growth of the cultural 

plant. Apart from parasitism and antibiosis Trichoderma sp. can compete with other fungi 

against nutrients and space. 

9 Experiments 

9.1 Experimental procedure 

The first step of the experiment which was undertaken by the author was to collect soil 

samples from different regions of Sri Lanka. The samples were taken from rambutan 

plantations and accordingly from soils straight underneath Annona trees. Afterwards 

Trichoderma spp. were isolated out of the soil samples. 

In a second step bio-assays were carried out to prove the effectiveness of the Trichoderma sp. 

against the pathogens, which has been isolated earlier from infected fruits. 

In another experiment, cross-inoculations were done to analyse whether diseased fruits of 

various species infect each other. 

Furthermore a sporulation experiment was undertaken. 

9.1.1 Laboratory set-up 

The most important thing while working in microbiological laboratories is to work under 

sterile conditions. Therefore the working places and all materials, as well as the tools and 

some chemicals have to be sterilized. 

The sterilization of most of the glassware, such as petri-dishes was done in a sterilization 

oven. The mode of action of the oven is hot dry air sterilization, in which at 180 °C in one 

hour’s time most organisms are killed. Hot moist air under pressure is the mode of action of 

the autoclaves (Figure 18). They are used for liquid sterilizations like media and water and 

other utilities such as funnels, beakers and pipettes as well as for things made out of 

polycarbonates like the pipette-tips. Autoclaves work at a temperature of 121 °C and a 

pressure of 1.1 kg/cm² and need only 15 minutes for sterilization. The reason why the 

23

autoclave is used for liquid sterilization is due to the lower temperature. Many chemicals are 

degenerating at a temperature more than 121 °C. 

Figure 18: Autoclave at ITI 

Figure 19: Laminar flow cabinet at ITI 

For the sterilization of the surface of the working place and the workers hands a 70 % ethanol 

solution is used. It is also used for the working utensils such as scalpel and forceps while 

working by dipping them into the alcohol solution and flaming them afterwards in the Bunsen 

burner flame. 

To sterilize the laminar flow cabinet or the incubator a so called fumigation should be done by 

filling a little amount of a 8 % formaldehyde dilution into a beaker or Petri-dish and keep it 

for more than a hour, when possible over night, in the closed incubator or laminar of flow 

cabinet. 

In all experiments which are described in this report the work was done under sterile 

conditions by using the above mentioned methods. 

The work with micro-organisms always takes place in so called laminar flow cabinet (Figure 

19). This is a half closed or manual closable glass cabin in which a constant air flow takes 

place and the air gets filtered to avoid contaminations. 

The medium which was used for all workings and experiments was a Potato-Dextrose-Agar 

(PDA) medium. The medium was prepared fresh, immediately before every single trial series. 

24

250 ml PDA was made up as follows: 

Material: 

• Bunsen burner 

• Screw cap bottle (300 ml) 

• Measuring cylinder (500 ml) 

• Funnel 

• Cloth for filtering 

• Beaker (500 ml) 

• Heating plate 

Chemicals: 

• 50 g potatoes 

• 5 g glucose 

• 5 g agar 

After washing, removing the skin and cutting into little quarters the potatoes have to boil in 

250 ml distilled water for 20 minutes. After boiling the solution has to be filtered through a 

funnel using a layer of muslin made up to a volume of 250 ml with distilled water. The 

solution is decanted into the screw cap bottle and the glucose and the sugar are added. After 

autoclaving the pH of the medium can be adjusted by adding 1.6 ml sterile tartaric acid per 

100 ml PDA to avoid bacterial growth. Lastly, the PDA is poured into sterile petri dishes 

(plates). In doing so the neck of the PDA-filled bottle should be held into the Bunsen burner 

flame once in a while to avoid contaminations. 250 ml 

PDA should be enough for 15 plates. 

During the experiments the cultures where stored in 

incubators which were set up to a temperature of 28 °C 

(Figure 20). The Trichoderma sp. cultures were put into an 

extra incubator. The reason for this is that Trichoderma sp. 

contaminate other cultures due to the very small and 

numerous conidia very easily. 

9.1.2 Soil sampling 

Figure 20: Incubator at ITI 

Soil samples from under rambutan and annona trees were taken in various regions of Sri 

Lanka. The samples were taken within the soil area which was covered by the crown of the 

tree. Out of this area four samples were taken randomly (Figure 21). Only the top soil to a 

deepness of 2 cm was taken and filled into plastic bags which were labelled and closed with a 

loose burl to guarantee an air exchange. 

25

Figure 21: Soil sampling in a rambutan plantation 

In this manner 16 soil samples were taken from 4 different rambutan plantations and 13 

samples from annona trees from 3 different areas. 7 of the “annona samples” were taken from 

Annona squamosa trees and 6 from Annona muricata trees; all the trees were standing in 

private gardens or little home gardens. 

Table 1 gives an overview of the soil samples and their place of sourcing. 

Table 1: An Overview of the soil samples which were taken 

District 

City 

Gampaha District Urapola 

Quantity soil samples of: 

rambutan A. squamosa A. muricata 

1 st plantation 4 

2 nd plantation 4 

3 rd plantation 4 

Date 

3. May 

Kegalle District Warakapola 4 8. June 

Anuradhapura Wijayapura 2 1 10. May 

District Medawachchiya 3 11. May 

Kegalle District Galagedera 1 12. May 

Matale District Matale 2 4 26. May 

9.1.3 Isolation of the bio-control agent 

To find the bio-control agent in the soil samples you have to see total number of fungi which 

are present in the soil. If Trichoderma sp. is present within the fungi an isolation can be done. 

26

The following materials and chemicals are needed to analyse a soil sample regarding the 

population of the fungi: 

Materials: 


• 2 flasks 

• Funnel carped with cotton wool 

• 6 closable tubes 

• Micropipette for 1 ml with 20 

pipette-tips 

• Petri-dishes filled with PDA 

Chemicals: 

• 10 g soil 

• 150 ml distilled water 

To analyse a soil sample a spore suspension has to be done before. Therefore 10 g of the soil 

sample was placed in a flask to which 10 ml distilled water was added and shaken carefully in 

a circular movement for 20 minutes. The solution was then filtered through a funnel with 

cotton wool into a second flask and in doing so the spore solution was obtained. 

Normally the spore concentration of the spore solution is too high and a dilution series has to 

be done. Therefore 1 ml of the spore solution was used with 9 ml distilled water to obtain a 

10 -1 dilution. This dilution procedure was repeated until a dilution series with the powers 10 -1 

to 10 -6 was obtained. 

0.1 ml of each dilution was spread on petridishes 

filled with PDA and incubated. These 

cultures (Figure 22) were observed every day for 

one week to check weather Trichoderma sp. is 

appearing somewhere. 

If the existence of the wanted fungus 

(Trichoderma sp.) was speculated an inspection 

by using the microscope was undertaken. If the 

identification was positive an isolation of the 

fungus to a pure culture was undertaken. 

Figure 22: Seven days old plate from 

a soil experiment 

27

9.1.4 Isolation of the pathogen 

Materials: 


• 4 beakers 

• Scalpel and forceps 

• 1 empty petri-dish 


• Pure PDA plates 

Chemicals: 

• 100 ml ethanol solution (70 %) 

• Sodium hypochlorite solution (5 %) 


For the isolation of the pathogen partially diseased fruit are required. The fruit should have 

enough healthy tissue. The fruit which were therefore bought either from local markets or 

collected during the field trips. 

From the fruit tissue squares were taken with the size of 1 cm² and the thickness of 3 mm. It 

was important that only 1/3 of the tissue was diseased. The tools which were necessary for 

this had to be sterilized after every cut (scalpel and forceps). The cuttings took place in an 

empty petri-dish. 

The pieces obtained were surface sterilized by dipping them 

for three minutes in a 5 % sodium hypochlorite solution. The 

solution was prepared immediately before the surface 

sterilization, because the effectiveness of the solution 

decelerates with running time. 

After that the pieces were washed twice by dipping them into 

Figure 23: Isoloation of the distilled water for three minutes to remove the rests of the 

pathogen on rambutan (five 

sterilization solution. In each case two washed fruit pieces 

days old plate) 

were put into one PDA plate and incubated. The mycelium of 

the pathogen covered the whole plate after a few days (Figure 23) and could be subcultivated. 

The pathogenicity of the isolated organism was proved using Koch’s postulates. Koch’s 

postulates can be summarised in four steps (FREDRICKS and RELMAN, 1996): 

• The organism must be found in all plants suffering from the disease, but not in 

healthy plants. 

• The organism must be isolated from a diseased plant and grown in pure culture. 

• The cultured organism should cause disease when introduced into a healthy plant. 

• The organism must be reisolated from the experimentally infected plant. 

28

If the Koch’s postulates are fulfilled it is proven that the isolated pathogen and the pathogen 

which causes the disease of the fruit are identical. 

9.1.5 Bio-Assays 

Materials: 


• Cork borer (number 5) 

• Spatula 

• Micropipette and pipette-tips 

For the spore suspension: 

• Small flask 

• Loop 


• Funnel with cotton wool 

Chemicals: 

• 150 ml Ethanol (70 %) 


The so called “bio-assays” are in vitro experiments and should give information about the 

activity of the bio-control agents (BC-agent) against the pathogens. Therefore several 

experimental series were undertaken, which respectively proved two BC-agents against four 

pathogens. 

The activity of the BC-agents was proved in petri-dishes filled with 15 ml PDA. Near the 

outer border of the Petri-dish four wells has been cut into the medium with a constant distance 

(Figure 24). The wells were cut with a cork 

borer number 5 (1 cm in diameter). Afterwards 

0.8 ml of a spore suspension of a BCagent 

was filled in each well. Following this 

a mycelia disk of the pathogen, which was 

cut with the same cork borer (No. 5) from a 

7 days old culture, was placed in the middle Figure 24: Preparation of a petri-dish for a bioassay 

of the petri-dish (Figure 24). 

The spore suspension was won out of 5 to 10 days old cultures of the BC-agents according to 

the time of full sporulation. Therefore 20 mm distilled water was filled into the culture plate 

and the upper layer of the fungus was scraped off the medium. This mix of distilled water and 

fungus tissue was filtered to receive a pure spore suspension of the BC-agent. For the controls 

the wells were filled with distilled water. In this manner five replications per pathogen took 

place, so that one experimental series consisted out of 60 culture plates. 

29

The effectivity of the BC-agent was established by doing daily measurements which recorded 

the diameter of the pathogen between the wells. If the diameter of the pathogen was getting 

smaller during the experimental time or the pathogen disappeared completely, the BC-agent 

had a good activity. If the BC-agent had no activity against the pathogen the diameter of the 

pathogen increased and the BC-agent could not develop. 

Each experimental series was incubated for 7 days at a temperature of 28 °C and daily 

observed (Figure 25 and Figure 26). 

Figure 25: BC-agent with a good (right) and a bad (left) effectiveness 

Figure 26: Experimental series of bio-assay after 2 (left) and 6 (right) days 

9.1.6 Cross-inoculation 

The cross-inoculation experiment is used as an examination if the pathogens which were 

isolated from the various fruits are host specific or if they are able to infect the other fruits 

with the same aggressiveness. 

30

The experiment was carried out on rambutan, sugar apple and soursop fruits and repeated. 

In every experimental trial four pathogens were tested on ten fruits and two different controls 

were undertaken, in which likewise ten fruits were used. So that in total 60 fruits were 

necessary for one experimental series. The soursops and sugar apples were bought from local 

markets in Colombo whereas the rambutan fruits were picked direct from a rambutan 

plantation. The experiments undertaken with rambutan were only done with fruits of the 

variety `Malwana Special´. 

At the beginning of the experiments the fruits had to be surface sterilized to avoid field 

inoculations. Therefore the fruits were dipped for five minutes into a 5 % sodium 

hypochlorite solution. After drying the fruits on air in the laminar flow cabinet one well was 

cut near the stem by using a cork borer (No. 5). It was important that the well was not cut too 

deep into the fruit at best only the skin should be removed. Than a seven day old mycelia disk 

of the pathogen was insert into the well. 

In the first control (control 1) the wells were filled with distilled water whereas the second 

control (control 2) consisted out of untreated fruits. 

All fruits where stored in self-made humidity chambers (Figure 27) during the experimental 

time of six days. An observation of the fruits was undertaken daily which gathered the disease 

spread in percent by using an infestation key (Figure 28 and Figure 29). 

Figure 27: Humidity chamber for the rambutan 

Cross-inoculation experiment 

Figure 28: Infestation key for crossinoculation 

experiment with sugar apple 

31

Figure 29: Experimental series of a cross-inoculation on Annona squamosa 

(day 2 to day 6) 

9.1.7 Sporulation experiment 

As mentioned in chapter 7 Botryodiplodia sp. under artificial conditions need a very long time 

to produce spores, which are crucial for the identification of the fungus. Therefore two 

different methods were tested which possibly affect the sporulation of Botryodiplodia sp. in 

consideration of its speed. 

The different Botryodiplodia sp. cultures were exposed to three different conditions. 

First all cultures were subcultured nine times and incubated for five days at 28 °C to obtain 

completely grown plates. Following the cultures were divided into three sets each with three 

plates. 

One set remained in the incubator, the second one was exposed to the daylight and the third 

one was exposed to daylight after a cold shock was admitted by storing the culture plates for 

seven days at 13 °C in a cold room. 

The experimental time was one month while the cultures were analysed regarding to spores 

twice a week. 

9.2 Results 

Trichoderma sp. found in the soil samples: 

In total twelve Trichoderma sp. could be isolated from six sources, which were different 

concerning their morphology and their development (Figure 30). The exact differences 

between the several isolates are found in the Annexes. Table 2 gives an overview of the 

isolated Trichoderma sp. regarding their source and the time they need to full sporulation of 

the plate. 

32

Table 2: An overview of the isolated Trichoderma sp. 

Code Source Fruit Full sporulation 

Tra I Urapola after 5 days 

Tra II Warakapola Rambutan after 5 days 

Tra III Warakapola 

after 5 days 

Tmu I Galagedera after 10 days 

Tmu II Galagedera after 6 days 

Annona 

Tmu III Galagedera after 6 days 

muricata 

Tmu IV Matale after 6 days 

Tmu V Wijayapura 

after 5 days 

Tsq I Matale after 6 days 

Tsq II Matale Annona after 5 days 

Tsq III Wijayapura squamosa after 5 days 

Tsq IV Medawachchiya 

after 6 days 

Figure 30: The twelve Trichoderma 

sp. Found in the soil samples 

Botryodiplodia sp. isolated from the fruits: 

Eight Botryodiplodia sp. could be isolated out of different fruits. Thereby three 

Botryodiplodia sp. were isolated from rambutan, three from Annona squamosa and one from 

Annona muricata fruits. Moreover one fungus was isolated out of a red skinned Annona 

squamosa. 

The fungi isolated out of the same fruits did not differ from each other. So it was assumed that 

it is the matter of the same species. 

Table 3 gives an overview of the isolated pathogens, in which only the four marked fungi 

(Figure 31) have been used for all the experiments. 

Table 3: An overview of the isolated pathogens 

Code Source Isolated from 

Bra I 

Market 

Bra II Warakapola (Plantation) 

rambutan 

Bra III 

Market 

Bmu I Market Annona muricata 

Bsq I Medawachchiya 

Bsq II 

Market 

Annona squamosa 

Bsq III 

Market 

Bsq IV Market Annona squamosa ´Red` 

Results of the bio-assays: 

Figure 31: The four Botryodiplodia 

sp. Isolates which were used for all 

experiments 

The bio-assay experiments have shown that three of the Trichoderma sp. isolates have a high, 

four a moderate and three a low activity against the pathogens, while three were observed as 

non effective (Table 4). 

33

Two of the Trichoderma sp. with a high effectiveness were isolated from rambutan 

plantations in Warakapola. The third of the high active ones was found in a soil sample taken 

from an Annona muricata tree in Wijayapura. The fungi which were isolated from soil 

samples taken from Annona squamosa trees showed the lowest activities against 

Botryodiplodia sp.. Only one of these fungi had a moderate effectiveness. 

Table 4 presents the results of the bio-assay, whereas “+++” identifies a good, “++” a 

moderate, “+” a low and “0” no effectiveness against the pathogens. 

Table 4: Results of the effectiveness of the isolated Trichoderma sp. 

Code Source Activity 

Tra I Urapola ++ 

Tra II Warakapola +++ 

Tra III Warakapola +++ 

Tsq I Matale 0 

Tsq II Matale + 

Tsq III Wijayapura ++ 

Tsq IV Medawachchiya 0 

Tmu I Galagedera ++ 

Tmu II Galagedera ++ 

Tmu III Galagedera 0 

Tmu IV Matale + 

Results of the cross-inoculation experiment: 

To interpret the results of the experiments with their replications as overall results T-tests 

were undertaken. In all tests the null hypothesis could be disproved. 

All pathogens have shown the same severity on the rambutan fruits after three days (Figure 

32). In the first two days the severity which was caused by the pathogen “Bmu I” was higher. 

Cross-inoculationNephelium lappaceum 

Severity [%] 

100 

80 

60 

40 

20 

0 

Day 1 Day 2 Day 3 Day 4 Day 5 

Time after inocculation [d] 

Bmu I Bsq IV Bra III Bsq III Control I Control II 

Figure 32: Results of the cross-inoculation on rambutan 

34

The evaluation of the cross-inoculation with Annona muricata showed that both controls have 

the same or a higher severity as the pathogens (Figure 33). On the fifth day of the experiment 

the severities of the pathogens Bmu I and Bsq III were higher than the ones of the others and 

of the controls. 

Cross-inoculationAnnona muricata 

Severity [%] 

100 

80 

60 

40 

20 

0 




Figure 33: Results of the cross-inoculation experiment on Annona muricata 

As it is seen in Figure 34, the standard deviations of the results of the experiments with 

Annona squamosa fruits are high. Thus the results allow no exact interpretation. 

Cross-inoculation Annona squamosa 

Severity [%] 

100 

80 

60 

40 

20 

0 




Figure 34: Result of the cross-inoculation on Annona squamosa 

35

Results of the sporulation experiment: 

Table 5 confirms results of the sporulation experiment: 

Table 5: Results of the sporulation experiment 

Pathogen 

Incubator 

Incubator and 

daylight 

Incubator, cold shock 

and daylight 

Bra II 

Sporulation after 24 

days 


days 


days 

Bmu I 


days 


days 

Bsq III 


days 


days 

Bsq IV 


days 


days 

The cold shock treatment of the cultures induced by all pathogens the longest sporulation 

duration, at Bsq IV a sporulation even failed to appear. The pathogens Bra II and Bsq II had 

the fastest sporulation with the daylight treatment without a cold shock. Bsq IV and Bmu I 

sporulated fastest in the incubator without any further treatment. But it took them with 24 

days the longest duration. 

36

10 Conclusion 

The results of the present study are showing that Trichoderma sp. are widely available in Sri 

Lanka and that some of those can be used as bio-control agents. SIVAKUMAR et al. (2000) 

isolated Trichoderma sp. from rambutan plantations and proved their antagonistic effects 

against Botryodiplodia theobromae as well. 

Only one of the bio-control agent (Bmu I) isolated from the surroundings of an Annona 

muricata tree was able to control the pathogen Bsq IV completely. This organism was 

isolated from a red skinned Annona squamosa. Consequently it seems that the Bio-control 

agent Bmu I is the most effective of all the BC-agents which were isolated for the 

experiments. 

The results of the cross-inoculation experiment show that cross infections between rambutan 

und annona fruits can appear both in the field and during transportation and storage. Thus it is 

to recommend that these fruits should not be transported or stored together. It also underlines 

the importance of the site selection of new plantations for both fruits. 

The isolated Trichoderma sp. should be tested in further studies in field experiments or in in 

vivo studies. For this, the BC-agent could be mixed into the soil or applied directly on the 

plant or fruit. 

According to BRIMMER and BOLAND (2003) some Trichoderma sp. can have negative effects 

on other useful organisms. This should be investigated in further studies as well. 

11 Acknowledgement 

I would like to thank the GTZ (Deutsche Gesellschaft für Technische Zusammenarbeit) for 

supporting my stay in Sri Lanka by giving me a scholarship and Prof. Dr. Joachim Heller 

from the University of Applied Sciences of Geisenheim for his support. 

Furthermore I would like to express my sincere gratitude to Dr. Hannah Jaenicke, director of 

ICUC and Dr. Shanthi Wilson Wijeratnam, Manager of the Post Harvest Technology Group 

of ITI for giving me the possibility to attend my BPS in Sri Lanka. 

I am indebted to the whole Post Harvest Technology Group and to the ICUC team for giving 

me a very warm and hearty welcome in a complete new surrounding. 

I would like to give sincere thankfulness to Mrs. Nimali Abeyratne, senior officer of ITI and 

to Miss. Yashoda Dharmatilaka for their great encouragement during my work at ITI. 

37

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40

13 Annexes 

Figure 35: Organisation structure of ITI (ITI, 2007) 

41

Figure 36: List of bio-control agents isolated 

Code Source Morphology Bio-control Activity Picture 

Bsq IV Bsq III Bra III Bmu I 

Tra I Urapola • Mycelial growth: 

(Madakotuwa) Covers the whole plate 

equal in 3 days after 

subculture. 

• Sporulation: 

Full sporulation after 5 

days; plate is dark green 

coloured. 

Tra II Etena velle • Mycelial growth: 

Covers the plate with 

thick white mycelia in 3 

days after subculture and 

starts turning green in the 

centre. 



days with domination in 

the centre and the outer 

area. 

Tra III Etena velle • Mycelial growth: 

Covers the whole plate 

with a very thick white 

layer after 4 days, the 

centre is yellowish-green 

coloured. After 6 days of 

incubation little black 

stone like balls are 

appearing. 

+ +++ +++ ++ 

++ +++ +++ +++ 

++ +++ +++ ++ 



days, but not homogeny 

coloured. 

Tsq I 

Matale 

(Home garden) 

• Mycelial growth: 

In 3 days after 

subculturing the whole 

plate is covered with a 

thin white mycelia, the 

media in the centre is 

turning into an orangeyellowish 

colour. 

0 0 0 0 



days but only less in the 

centre and the outer area. 

42

Tsq II 

Matale 

(Homegarden) 

• Mycelial growth: 

Covers the full plate in 4 

days after subculturing. 

The colour is already light 

green. 

0 ++ ++ + 



days, dark green colour. 

Tsq III Wijayapura • Mycelial growth: 

Mycelium is growing like 

many little dots which are 

spread on the plate. After 

5 days the whole plate is 

covered with this little 

“mycelia dots”. 


Complete sporulation of 

the mycelia after 7 days. 

Tsq IV Medawachchiya • Mycelial growth: 

Mycelium is growing 

very heterogenic. After 4 

days the complete plate is 

covered. But the mycelia 

layer is not out of one 

piece. Spot like in the 

outer part of the plate. It 

has a bad smell. 

+ +++ +++ ++ 

0 0 0 0 


Sporulation after 6 days. 

Tmu I Galagedera • Mycelial growth: 

Whole plate is covered in 

3 days after subculture 

with snow-white mycelia. 

After 8 days the mycelia 

starts to turn into a very 

light green. 

++ ++ + ++ 



days. 

Tmu II Galagedera • Mycelial growth: 

The plate is covered with 

white mycelia after 3 

days. The mycelia around 

the subcultered piece is 

turning dark green after 4 

days while the rest stays 

white till it turns light 

green after 5 days. 

++ ++ +++ ++ 


Sporulation of the full 

plate after 6 days after 

subculture 

43

Tmu III Galagedera • Mycelial growth: 

Covers the whole plate in 

4 days. Some parts of the 

mycelia are turning the 

colour into dark-green 

and some are staying 

white with a very thick 

mycelium-layer. 



days, the white parts are 

getting a light greenyellowish 

colour. 

Tmu IV Matale • Mycelial growth: 

After 4 days the whole 

plate is covered with are 

white mycelia, except the 

centre which green 

coloured. The surface of 

the mycelia is covered 

with many little craters. 

0 0 0 0 

++ ++ + 0 



days, when the complete 

mycelia changed into a 

dark green colour. 

Tmu V Wijayapura • Mycelial growth: 

The mycelium never 

covers the whole plate 

and is not growing in a 

round way. After 4 days 

the mycelia changes into a 

light green colour. 

+++ ++ +++ +++ 


After 5 days with a very 

dark green colour. 

44

Botryodiplodia sp. - Crops for the Future

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