Botryodiplodia sp. - Crops for the Future
Botryodiplodia sp. - Crops for the Future
Botryodiplodia sp. - Crops for the Future
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Control of Post Harvest Disease<br />
(<strong>Botryodiplodia</strong> <strong>sp</strong>.) of Rambutan and Annona<br />
Species by Using a Bio-Control Agent<br />
(Trichoderma <strong>sp</strong>.)<br />
Robert Kunz<br />
June 2007
List of Contents<br />
List of Contents .......................................................................................................................... 1<br />
List of Figures ............................................................................................................................ 2<br />
List of Tables.............................................................................................................................. 3<br />
List of Acronyms........................................................................................................................ 3<br />
1 Summary ................................................................................................................................. 4<br />
2 Introduction ............................................................................................................................. 5<br />
3 Sri Lanka ................................................................................................................................. 6<br />
3.1 Geography ........................................................................................................................ 6<br />
3.2 Climate ............................................................................................................................. 8<br />
3.3 Socio-economics .............................................................................................................. 8<br />
4 The host institutes.................................................................................................................... 9<br />
4.1 International Centre <strong>for</strong> Underutilised <strong>Crops</strong> (ICUC)...................................................... 9<br />
4.2 Industrial Technology Institute (ITI)................................................................................ 9<br />
5 Reason <strong>for</strong> research project ................................................................................................... 10<br />
6 The plants .............................................................................................................................. 11<br />
6.1 Rambutan (Nephelium lappaceum)................................................................................ 11<br />
6.1.1 Description of <strong>the</strong> plant/fruit ................................................................................... 11<br />
6.1.2 Main diseases .......................................................................................................... 13<br />
6.1.3 Economics in Sri Lanka .......................................................................................... 13<br />
6.2 Annona (Annona <strong>sp</strong>.)...................................................................................................... 13<br />
6.2.1 Description of <strong>the</strong> plant/fruit ................................................................................... 14<br />
6.2.1.1 Annona cherimola ................................................................................................ 14<br />
6.2.1.2 Annona muricata .................................................................................................. 16<br />
6.2.1.3 Annona squamosa................................................................................................. 17<br />
6.2.1.4 Annona reticulata................................................................................................. 18<br />
6.2.2 Main diseases .......................................................................................................... 19<br />
6.2.3 Economics in Sri Lanka .......................................................................................... 19<br />
7 The disease (<strong>Botryodiplodia</strong> <strong>sp</strong>.)........................................................................................... 19<br />
7.1 Taxonomy....................................................................................................................... 19<br />
7.2 Disease pattern ............................................................................................................... 21<br />
8 Bio-control agent (Trichoderma <strong>sp</strong>.)..................................................................................... 21<br />
8.1 Taxonomy....................................................................................................................... 21<br />
8.2 The role of mycoparasitism............................................................................................ 22<br />
9 Experiments........................................................................................................................... 23<br />
9.1 Experimental procedure ................................................................................................. 23<br />
9.1.1 Laboratory set-up ........................................................................................................ 23<br />
9.1.2 Soil sampling........................................................................................................... 25<br />
9.1.3 Isolation of <strong>the</strong> bio-control agent ............................................................................ 26<br />
9.1.4 Isolation of <strong>the</strong> pathogen ......................................................................................... 28<br />
9.1.5 Bio-Assays............................................................................................................... 29<br />
9.1.6 Cross-inoculation..................................................................................................... 30<br />
9.1.7 Sporulation experiment ........................................................................................... 32<br />
9.2 Results ............................................................................................................................ 32<br />
10 Conclusion........................................................................................................................... 37<br />
11 Acknowledgement............................................................................................................... 37<br />
12 References ........................................................................................................................... 38<br />
13 Annexes............................................................................................................................... 41<br />
1
List of Figures<br />
Figure 1: The Sri Lankan Flag ..................................................................................... .... 6<br />
Figure 2: The Sri Lankan coat of arms ........................................................................ ..... 6<br />
Figure 3: Map of Sri Lanka ......................................................................................... ..... 7<br />
Figure 4: Different shapes of <strong>the</strong> rambutan fruit: 1) oval, 2) ovoid and 3)<br />
Ellipsoid......................................................................................................... .. 12<br />
Figure 5:<br />
The most important rambutan varieties in Sri Lanka: `Malwana Special´,<br />
`Malaysian Yellow´ and `Malaysian Red’ .................................................... .. 12<br />
Figure 6: Annona cherimola ......................................................................................... .. 15<br />
Figure 7: Annona muricata tree .................................................................................... .. 16<br />
Figure 8: Annona muricata ........................................................................................... .. 16<br />
Figure 9: Annona squamosa ......................................................................................... .. 18<br />
Figure 10: A red skinned Annona squamosa fruit ......................................................... . . 18<br />
Figure 11: Annona reticulate .......................................................................................... .. 18<br />
Figure 12: Mature and immature <strong>sp</strong>ores of <strong>Botryodiplodia</strong> <strong>sp</strong>. ...................................... .. 20<br />
Figure 13:<br />
Petri-dish with <strong>Botryodiplodia</strong> <strong>sp</strong>. after seven days (left) and after<br />
four Weeks (right) ....................................................................................... .. 20<br />
Figure 14: A. muricata fruit infected with diplodia rot................................................... .. 21<br />
Figure 15: Phialides of Trichoderma <strong>sp</strong>.......................................................................... .. 22<br />
Figure 16: Petri-dish with a six day old Trichoderma <strong>sp</strong>. Colony.................................. .. 22<br />
Figure 17: Trichoderma harzianum parasitizes Pythium ultimum.................................. .. 22<br />
Figure 18: Autoclave at ITI............................................................................................. .. 24<br />
Figure 19: Laminar of flow at ITI................................................................................... .. 24<br />
Figure 20: Incubator at ITI.............................................................................................. .. 25<br />
Figure 21: Soil sampling in a rambutan plantation......................................................... .. 26<br />
Figure 22: Seven days old plate from a soil experiment................................................. .. 27<br />
Figure 23: Isolation of <strong>the</strong> pathogen on rambutan (five days old plate .......................... .. 28<br />
Figure 24: Preparation of a Petri-dish <strong>for</strong> a bio-assay .................................................... .. 29<br />
Figure 25: BC-agent with a good (right) and with a bad (leftight) effectiveness ........... .. 30<br />
Figure 26: Experimental series of a bio-assay after two (left) and six (right) days........ .. 30<br />
Figure 27: Humidity chamber of <strong>the</strong> rambutan cross-inoculation experiment ............... .. 31<br />
Figure 28: Infestation key <strong>for</strong> <strong>the</strong> cross-inoculation experiment with sugar apple......... .. 31<br />
Figure 29: Experimental series of a cross-inoculation on A. squamosa<br />
(day 2 to day 6).............................................................................................. .. 32<br />
Figure 30: The twelve Trichoderma <strong>sp</strong>. found in <strong>the</strong> soil samples................................. .. 33<br />
Figure 31:<br />
The four <strong>Botryodiplodia</strong> <strong>sp</strong>. isolates which were used <strong>for</strong> all<br />
Experiments................................................................................................... .. 33<br />
Figure 32: Results of <strong>the</strong> cross-inoculation on rambutan ............................................... .. 34<br />
Figure 33: Results of <strong>the</strong> cross-inoculation on Annona muricata................................... .. 35<br />
Figure 34: Results of <strong>the</strong> cross-inoculation on Annona squamosa................................. .. 35<br />
Figure 35: Organisation structure of <strong>the</strong> ITI................................................................... .. 41<br />
Figure 36: List of <strong>the</strong> bio-control agents isolated ........................................................... .. 42<br />
2
List of Tables<br />
Table 1: An Overview of <strong>the</strong> soil samples which were taken……………………………….. 26<br />
Table 2: An overview of <strong>the</strong> isolated Trichoderma <strong>sp</strong>………………………………………. 33<br />
Table 3: An overview of <strong>the</strong> isolated pathogens…………………………………………….. 33<br />
Table 4: Results of <strong>the</strong> effectiveness of <strong>the</strong> isolated Trichoderma <strong>sp</strong>……………………….. 34<br />
Table 5: Results of <strong>the</strong> <strong>sp</strong>orulation experiment……………………………………………… 36<br />
List of Acronyms<br />
BC-agent - Bio-control agent<br />
BPS - Beruf<strong>sp</strong>raktisches Projektsemester<br />
CGIAR - Consultative Group on International Agricultural Research<br />
CISIR - Ceylon Institute of Scientific and Industrial Research<br />
DOASL - Department of Agriculture, Sri Lanka<br />
GTZ - Deutsche Gesellschaft für Technische Zusammenarbeit<br />
HIV - Human immunodeficiency virus<br />
ICUC - The International Centre <strong>for</strong> Underutilised <strong>Crops</strong><br />
IPGRI - International Plant Genetic Resources Institute<br />
ITI - Industrial Technology Institute<br />
IWMI - International Water Management Institute<br />
PDA - Potato Dextrose Agar<br />
UK - United Kingdom<br />
3
1 Summary<br />
Experiments were undertaken by <strong>the</strong> Industrial Technology Institute (ITI) in cooperation with<br />
<strong>the</strong> International Centre <strong>for</strong> Underutilised <strong>Crops</strong> (ICUC) to prove <strong>the</strong> availability of<br />
Trichoderma <strong>sp</strong>. in Sri Lanka and <strong>the</strong>ir effectiveness as Bio-control agents (BC-agents)<br />
against <strong>Botryodiplodia</strong> <strong>sp</strong>.<br />
The Trichoderma <strong>sp</strong>. used in <strong>the</strong> experiments were isolated from soil samples taken from<br />
several locations in Sri Lanka and <strong>the</strong> pathogens were isolated from diseased fruits of Annona<br />
squamosa, Annona muricata and rambutan (Nephelium lappaceum).<br />
Bio-assays were undertaken to prove <strong>the</strong> effectiveness of <strong>the</strong> BC-agents. In addition crossinoculation<br />
experiments were done to see whe<strong>the</strong>r <strong>the</strong> pathogens of various fruits infect each<br />
o<strong>the</strong>r. Fur<strong>the</strong>rmore a <strong>sp</strong>orulation experiment was attempted to check if a reduction of <strong>the</strong><br />
<strong>sp</strong>orulation time of <strong>Botryodiplodia</strong> <strong>sp</strong>. under <strong>sp</strong>ecial treatments (cold shock and daylight) is<br />
possible.<br />
Following results of <strong>the</strong> experiments are shown:<br />
Isolates:<br />
Twelve isolates of Trichoderma were obtained from six different soil-sources and eight<br />
isolates of <strong>the</strong> pathogen from diseased fruits. One pathogen from each fruit was taken <strong>for</strong><br />
fur<strong>the</strong>r experiments.<br />
Bio-assay:<br />
The bio-assay experiment showed that three of <strong>the</strong> Trichoderma <strong>sp</strong>. isolates have a high, four<br />
have moderate and three have low Bio-control activity while three isolates observed to be non<br />
effective. Two of <strong>the</strong> Trichoderma strains with a high BC-activity were isolated from<br />
rambutan orchards in Warakapola and <strong>the</strong> third was found in a soil sample, taken from <strong>the</strong><br />
surrounding area of an Annona muricata tree in Wijayapura.<br />
None of <strong>the</strong> isolates taken from Annona squamosa sources showed high BC-activity, while<br />
one was moderately effective against <strong>the</strong> pathogens.<br />
Cross-inoculation experiment:<br />
The pathogens which were cross-inoculated to <strong>the</strong> rambutan fruits showed an equal severity<br />
after <strong>the</strong> third day. On <strong>the</strong> first two days <strong>the</strong> severity that was caused by <strong>the</strong> pathogen isolated<br />
from Annona muricata was a little higher.<br />
4
The cross-inoculation experiment with Annona muricata fruits did not lead to satisfactory<br />
results. The reason being that <strong>the</strong> severity of infection in <strong>the</strong> two controls were <strong>the</strong> same or<br />
higher to <strong>the</strong> severity caused by <strong>the</strong> pathogens. A statistically significant higher severity<br />
between pathogen taken from Annona muricata and <strong>the</strong> one taken from Annona squamosa<br />
compared to <strong>the</strong> o<strong>the</strong>rs can be seen at <strong>the</strong> fifth day.<br />
The variability in <strong>the</strong> cross-inoculation experiment with Annona squamosa fruits was high;<br />
never<strong>the</strong>less we can conclude that <strong>the</strong> pathogens of <strong>the</strong> various fruits do infect <strong>the</strong> o<strong>the</strong>rs.<br />
Sporulation experiment:<br />
The treatment with a cold shock resulted in <strong>the</strong> longest <strong>sp</strong>orulation times of 26 days with<br />
re<strong>sp</strong>ect to all pathogens which were observed; <strong>the</strong> pathogen isolated from a red skinned<br />
Annona squamosa which did not <strong>sp</strong>orulate during <strong>the</strong> experimental period. The pathogens<br />
which were isolated from rambutan and Annona squamosa <strong>sp</strong>orulated fastest (15 days) under<br />
<strong>the</strong> daylight treatment. The pathogens isolated from <strong>the</strong> red skinned Annona squamosa and<br />
from Annona muricata <strong>sp</strong>orulated fastest (24 days) in <strong>the</strong> incubator without any treatment.<br />
The two collaborative Institutes are described in this report as well as <strong>the</strong> country. Characteristics<br />
of both fruits used <strong>for</strong> <strong>the</strong> experiments and o<strong>the</strong>r important Annona <strong>sp</strong>ecies (A. cherimola and A.<br />
reticulata) as well as <strong>the</strong> two fungi are also described.<br />
2 Introduction<br />
The background <strong>for</strong> <strong>the</strong> research presented here was <strong>the</strong> “Beruf<strong>sp</strong>raktisches Projeksemester”<br />
(BPS) programme which has to be done as a practical part of <strong>the</strong> author’s studies towards a<br />
Bachelor of Science degree in Horticulture-Management.<br />
The work was done in two institutes in Sri Lanka: The International Centre <strong>for</strong> Underutilised<br />
<strong>Crops</strong> (ICUC) which has announced <strong>the</strong> internship and <strong>the</strong> Industrial Technology Institute<br />
(ITI) which was <strong>the</strong> main working place <strong>for</strong> <strong>the</strong> experimental trails.<br />
The Intern was involved in a project of ITI and conducted several experiments which are<br />
described on <strong>the</strong> following pages.<br />
The main task of <strong>the</strong> intern was to prove antagonistic effects of fungus (Trichoderma <strong>sp</strong>.)<br />
against a fungal pathogen (<strong>Botryodiplodia</strong> <strong>sp</strong>.).<br />
5
3 Sri Lanka<br />
The Democratic Socialist Republic of Sri Lanka is an insular state in <strong>the</strong> Indian Ocean and<br />
was earlier known under <strong>the</strong> name Ceylon. Sri Lanka is a member state of <strong>the</strong> Commonwealth<br />
with <strong>the</strong> seat of <strong>the</strong> government is Sri Jayewardenepura and <strong>the</strong> capital as well as <strong>the</strong> biggest<br />
city is Colombo.<br />
The following Figures (1 and 2) are showing <strong>the</strong> flag and <strong>the</strong> coat of arms of Sri Lanka.<br />
Figure 1: The Sri Lankan flag (MY WORLD GUIDE, 2007)<br />
Figure 2: The Sri Lankan coat of arms (MY WORLD GUIDE, 2007)<br />
3.1 Geography<br />
Sri Lanka is a tropical island lying close to <strong>the</strong> sou<strong>the</strong>rn tip of India and near <strong>the</strong> Equator<br />
between 6° and 10° nor<strong>the</strong>rn latitude and between 79° and 82° eastern longitude. It is parted<br />
from India through <strong>the</strong> Palk Strait and <strong>the</strong> Gulf of Mannar. The coral islands of <strong>the</strong> Adam’s<br />
Bridge represent an incoherent connection of <strong>the</strong> north-western Sri Lanka and <strong>the</strong> Indian state<br />
Tamil Nadu.<br />
6
The longest destination from <strong>the</strong> north to <strong>the</strong> south is about 440 km and <strong>the</strong> widest part of <strong>the</strong><br />
island measures around 220 km. The total expanse of <strong>the</strong> country is 65,610 km² and <strong>the</strong><br />
highest elevation of <strong>the</strong> central highlands is <strong>the</strong> Pidurutalagala with 2524 m above sea level.<br />
Sri Lanka is commonly divided in three different landscapes: <strong>the</strong> central highlands, <strong>the</strong><br />
lowland plains and <strong>the</strong> coastal areas (Figure 3).<br />
Figure 3: Map of Sri Lanka (THE UNIVERSITY OF TEXAS, 2000)<br />
7
3.2 Climate<br />
The climate of Sri Lanka is tropical with different precipitation rates which accrue due to <strong>the</strong><br />
effects of <strong>the</strong> monsoons. While <strong>the</strong> southwest of <strong>the</strong> country is humid with two precipitation<br />
maxima in May and October, <strong>the</strong> sou<strong>the</strong>ast monsoon brings only little rainfall on <strong>the</strong> nor<strong>the</strong>ast<br />
and east coast because this area is in <strong>the</strong> lee of <strong>the</strong> central mountains. In <strong>the</strong>se areas most<br />
precipitation falls due to <strong>the</strong> nor<strong>the</strong>ast monsoon in November and December.<br />
The mean annual temperature is 22.2 °C in Kandy, 27.8 °C in Colombo and 33 °C in<br />
Tricomalee.<br />
The natural vegetation of Sri Lanka varies according to climatic zone and elevation. Dense<br />
evergreen rain <strong>for</strong>ests are found in <strong>the</strong> south-western lowlands. In <strong>the</strong> central highlands,<br />
evergreen mountain <strong>for</strong>ests are inter<strong>sp</strong>ersed with grasslands. The drier evergreen <strong>for</strong>ests in <strong>the</strong><br />
north and east contain trees such as ebony and satinwood. Thorn <strong>for</strong>ests and drought-resistant<br />
shrubs prevail in <strong>the</strong> driest areas. Along <strong>the</strong> coast, mangrove <strong>for</strong>ests border lagoons and river<br />
estuaries.<br />
3.3 Socio-economics<br />
The population of Sri Lanka is about 20 million (AUSWAERTIGES AMT, 2007) and ethnically<br />
heterogeneous. 74.6 % of <strong>the</strong> population are Singhalese, 18.1 % are Tamil, 7 % are Arabians<br />
and 0.3 % are o<strong>the</strong>rs.<br />
The Tamils were deployed from <strong>the</strong> British during <strong>the</strong> colonial time mainly as workers at tea<br />
plantations or as workers in <strong>the</strong> administration which led to a favouritism of <strong>the</strong> Tamil<br />
population compared to <strong>the</strong> Singhalese population. This caused, after <strong>the</strong> independency of <strong>the</strong><br />
country in 1948, great antipathies between <strong>the</strong> two population groups, whereupon <strong>the</strong> Tamil<br />
population of <strong>the</strong> north and <strong>the</strong> east tried first peacefully <strong>the</strong>n with <strong>for</strong>ce of arms to create<br />
<strong>the</strong>ir own state. This was <strong>the</strong> beginning of <strong>the</strong> civil war which still is a continuous armed<br />
conflict.<br />
Sri Lanka is also a country of religious diversity. Most of <strong>the</strong> population are Buddhists (69.3<br />
%) which are mainly Singhalese people. 15.5 % are Hindus where most of <strong>the</strong>m are Tamil<br />
people followed by Christians (Singhalese and Tamils) with 7.6 % of <strong>the</strong> population and last<br />
Muslims with 7.5 %.<br />
According to <strong>the</strong> AUSWAERTIGE AMT (2007) <strong>the</strong> economy of Sri Lanka has risen in <strong>the</strong> year<br />
2005 about a value of 5.3 % and <strong>for</strong> <strong>the</strong> year 2006 again a high economic growth was<br />
expected. The economical development of <strong>the</strong> country shows high regional differences. The<br />
8
economic centre is <strong>the</strong> region around Colombo which achieves nearly half of <strong>the</strong> total<br />
economic per<strong>for</strong>mance.<br />
The economic change of structure shows that <strong>the</strong> agricultural sector (tea, rubber and coconut)<br />
was dominating once, but at present only makes up 18 % of <strong>the</strong> economical overall<br />
per<strong>for</strong>mance. 26 % are contributing by industry, e<strong>sp</strong>ecially <strong>the</strong> textile sector. The service<br />
sector makes up 55 % of <strong>the</strong> economical per<strong>for</strong>mance e<strong>sp</strong>ecially <strong>the</strong> tourist sector.<br />
In <strong>the</strong> agricultural sector <strong>the</strong> tea industry has a <strong>sp</strong>ecial relevance. The exports of tea yield<br />
about 20 % of <strong>the</strong> <strong>for</strong>eign exchange proceeds. The tourist sector has lost on relevance due to<br />
<strong>the</strong> Tsunami in 2004 and due to <strong>the</strong> upcoming ethnic conflict.<br />
4 The host institutes<br />
4.1 International Centre <strong>for</strong> Underutilised <strong>Crops</strong> (ICUC)<br />
The International Centre <strong>for</strong> Underutilised <strong>Crops</strong> (ICUC) is a global research, development<br />
and training organisation which was established in 1992. It is a Partner Organisation to <strong>the</strong><br />
Consultative Group on International Agricultural Research (CGIAR).<br />
Since 2005 ICUC is located in <strong>the</strong> complex of buildings of <strong>the</strong> International Water<br />
Management Institute (IWMI) in Sri Lanka (Colombo) and is working <strong>the</strong>re independently.<br />
To describe <strong>the</strong> activities of ICUC <strong>the</strong> term “underutilised crops” has to be defined first:<br />
According to ICUC (2006) underutilised crops “are plant <strong>sp</strong>ecies which are used traditionally<br />
<strong>for</strong> <strong>the</strong>ir food, fibre, fodder, oil or medicinal properties. They have an under-exploited<br />
potential to contribute to food security, nutrition, health, income generation and environmental<br />
services.”<br />
The mission of <strong>the</strong> institute is “to promote <strong>the</strong> use of underutilised crops <strong>for</strong> <strong>the</strong> benefit of humankind<br />
and <strong>the</strong> environment to reduce poverty and suffering through <strong>the</strong> improvement and<br />
promotion of underutilised crops.”<br />
ICUC works in collaboration with many national and international partners and acts as a<br />
knowledge hub <strong>for</strong> tropical, sub-tropical and temperate plant development.<br />
The Industrial Technology Institute (ITI) is one of <strong>the</strong> partners ICUC is working with.<br />
4.2 Industrial Technology Institute (ITI)<br />
The Industrial Technology Institute (ITI) is a Statutory Board, which came into existence as<br />
successor to <strong>the</strong> Ceylon Institute of Scientific and Industrial Research (CISIR) on 1998. The<br />
ITI functions within <strong>the</strong> purview of <strong>the</strong> Ministry of Science and Technology. ITI provides<br />
9
valuable consultations in industrial, scientific and technological areas and offers <strong>the</strong> major<br />
analytic services which are necessary <strong>for</strong> <strong>the</strong> public and private sector industry. Among o<strong>the</strong>r<br />
things food- and product assays, soil- and water analyses and functional tests are carried out at<br />
ITI (ITI, 2001).<br />
In 1991 <strong>the</strong> research and development policies and strategies were re<strong>for</strong>mulated as <strong>the</strong><br />
demands of industry and o<strong>the</strong>r private sectors have changed. This new policy underlined <strong>the</strong><br />
need to be market oriented and demand driven with re<strong>sp</strong>ect to its research and development<br />
and service function with a view to being self-financing.<br />
Today <strong>the</strong> ITI maintains its premier role as an interdisciplinary research institute in Sri Lanka<br />
dedicated to <strong>the</strong> promotion of industrial development through research, consultancy and o<strong>the</strong>r<br />
technical services and stands committed to serve <strong>the</strong> nation to meet <strong>the</strong> challenges of <strong>the</strong><br />
future (ITI, 2007).<br />
The ITI has about 355 staff and is structured in six divisions, which consist of <strong>sp</strong>ecialized<br />
groups (Annexes, Figure 34). The author main working place was located in <strong>the</strong> Post Harvest<br />
Technology Group of <strong>the</strong> Agro and Food Technology Division. The group consists out of<br />
eight people.<br />
5 Reason <strong>for</strong> research project<br />
Recent studies have shown <strong>the</strong> importance of products of underutilised fruits, such as jams,<br />
juices and candied fruits with regard to nutrition, income generation and poverty reduction of<br />
small scale entrepreneurs in developing countries (AZAM-ALI, 2003). Underutilised tropical<br />
fruits such as rambutan (Nephelium lappaceum) and annona (Annona <strong>sp</strong>.) provide important<br />
contributions to small-holder livelihoods. However, heavy post-harvest losses significantly<br />
reduce <strong>the</strong> full potential <strong>for</strong> income generation.<br />
The Post Harvest Technology Group of ITI is working on a project which develops biological<br />
non chemical methods <strong>for</strong> control of post harvest diseases of tropical fruits such as mango,<br />
banana and papaya as well as working toge<strong>the</strong>r with ICUC on promising underutilised <strong>sp</strong>ecies<br />
such as rambutan and annona. The project presented here was part of this research program.<br />
The stem-end rot is caused by <strong>the</strong> fungus <strong>Botryodiplodia</strong> <strong>sp</strong>. and is one of <strong>the</strong> major post<br />
harvest diseases on many fruits (GUPTA et al., 1999, PLOETZ, 2003 and ALAM et al., 2001).<br />
Among o<strong>the</strong>rs it counts to <strong>the</strong> most important pre- and post harvest diseases on rambutan<br />
(SIVAKUMAR et al., 1997) and annona <strong>sp</strong>ecies (DE Q. PINTO, 2005). The use of <strong>the</strong> mycoparasitic<br />
fungus Trichoderma <strong>sp</strong>. as a bio-control agent represents an alternative plant protection<br />
10
measure to chemical treatments. The effectiveness of Trichoderma <strong>sp</strong>. against certain fungi<br />
has been proven in a number of experiments (SIVAKUMAR et al., 2000, BARBOSA et al., 2001<br />
and WANTOCH-REKOWSKI, 2004).<br />
The following three points were <strong>the</strong> main task of <strong>the</strong> study which is described in this report:<br />
• To establish whe<strong>the</strong>r bio-control agents are widely available in Sri Lanka.<br />
• To prove <strong>the</strong> efficacy of <strong>the</strong> bio-control agents against <strong>the</strong> pathogen.<br />
• To prove whe<strong>the</strong>r diseased fruits of various <strong>sp</strong>ecies infect each o<strong>the</strong>r.<br />
6 The plants<br />
6.1 Rambutan (Nephelium lappaceum)<br />
Rambutan belongs to <strong>the</strong> family of Sapindaceae. Its common botanical name is Nephelium<br />
lappaceum, but <strong>the</strong>re are two o<strong>the</strong>r synonym names: Euphoria nephelium and Dimocarpus<br />
crinita (MORTON, 1987a). The family Sapindaceae includes many fruit <strong>sp</strong>ecies but only<br />
rambutan, litchi (Litchi chinensis) and longan (Euphoria longana) have commercial importance<br />
(REHM and ESPIG, 1984).<br />
The origin of <strong>the</strong> fruit is Malaysia and it is mostly cultivated in Sou<strong>the</strong>ast Asia. A few<br />
rambutan trees are also grown in <strong>the</strong> costal lowlands of Colombia, Ecuador, Honduras, Costa<br />
Rica, Trinidad and Cuba. Some trees are also cultivated in Australia. (LAKSMI et al., 1987).<br />
Rambutan flourishes best in humid tropical regions with well distributed rainfall (2500-3000<br />
mm) and in deep soils with high organic matter. The plant grows from sea level to 500 m<br />
height and needs temperatures over 10 °C. The dry season should not last more than three<br />
months (REHM and ESPIG, 1984). According to MORTON (1987a) <strong>the</strong> Oriental Mindora region<br />
of <strong>the</strong> Philippines with an average temperature of about 27.3 °C, a relative humidity of 82 %<br />
and about 165 rainy days is <strong>the</strong> ideal environment.<br />
The name rambutan comes from <strong>the</strong> Malayan word “rambut” and means hairy, which<br />
correlates with <strong>the</strong> appearance of <strong>the</strong> fruit.<br />
6.1.1 Description of <strong>the</strong> plant/fruit<br />
The height of <strong>the</strong> rambutan tree varies between <strong>the</strong> different varieties and <strong>the</strong> method of<br />
cultivation from 8 to 25 m (BAERTELS, 1990, FRANKE, 1994 and MORTON, 1987a). The<br />
evergreen tree has alternate pinnate 7 to 30 cm long leaves with 1 to 4 pairs of leaflets. The<br />
leaflets are elliptic to oblong, pale shining with a dark-green colour.<br />
11
Rambutan trees are ei<strong>the</strong>r dioecious or monoecious with male or hermaphrodite flowers and<br />
need cross pollination <strong>for</strong> fruit set. The petal-less flowers are very incon<strong>sp</strong>icuous because of<br />
<strong>the</strong>ir tiny size (2.5 to 5 mm) and <strong>the</strong>ir greenish colour. They are growing on long shafted and<br />
much branched, to 30 cm long panicles and have a sweet smelling fragrance.<br />
The fruit is maturing in 15 to 18 weeks after flowering and gets an ovoid, round or ellipsoid<br />
shape with a pinkish-red, bright- or deep-red, orange-red, maroon or dark-purple, yellowishred<br />
or yellowish colour (Figure 4). The size of <strong>the</strong> rambutan fruit varies from 3.4 to 8 cm<br />
(BAERTELS, 1990, FRANKE, 1994 and MORTON, 1987a). The fruit has a thin parchment like<br />
rind with tubercles from each of which extends a soft <strong>sp</strong>ine. These hairy <strong>sp</strong>ines are 0.5 to 2<br />
cm long and have a red, pinkish or greenish-yellow colour (Figure 5).<br />
Figure 4: Different shapes of <strong>the</strong> rambutan fruit: 1) oval, 2) ovoid and 3) ellipsoid (IPGRI, 2003)<br />
Figure 5: The most important rambutan varieties in Sri Lanka: `Malwana Special´, `Malaysian<br />
Yellow´ and `Malaysian Red<br />
The white or rose-tinted and translucent 0.4 to 0.8 cm thick flesh is very juicy and covers one<br />
ovoid to oblong almond-shaped dark seed which is 2.5 to 3.5 cm long and 1 to 1.5 cm wide.<br />
Rambutan fruits have an exotic aroma which results out of <strong>the</strong> interaction of fruity-sweet and<br />
fatty-green odours, with <strong>the</strong> possible contribution of civet-like sweetish, <strong>sp</strong>icy and woody<br />
notes (ONG, ACREE and LAVIN, 1998).<br />
12
6.1.2 Main diseases<br />
The most important yield reducing factor in rambutan plantations are birds and flying foxes<br />
and some o<strong>the</strong>r non flying mammals that consume many of <strong>the</strong> fruits (MORTON, 1987a).<br />
According to MORTON (1987a) <strong>the</strong> major pests are leaf eating insects like <strong>the</strong> mealy bug<br />
(Pseudococcus lilacinus) and <strong>the</strong> giant bug (Tessaratoma longicorne). The Oriental fruit fly<br />
attacks very ripe fruits and counts, according to FRANKE (1994), to <strong>the</strong> most important pests.<br />
There are several pathogens that attack <strong>the</strong> fruit and cause rotting under warm and moist<br />
conditions, like <strong>Botryodiplodia</strong> <strong>sp</strong>. which was mentioned be<strong>for</strong>e. These fungi are important<br />
post harvest diseases.<br />
Oidium <strong>sp</strong>. Phomopsis <strong>sp</strong>. and o<strong>the</strong>r soil born fungi maintain damage on foliage and o<strong>the</strong>r<br />
parts of <strong>the</strong> tree. Fomes lignosus is causing <strong>the</strong> stem canker in <strong>the</strong> Philippines and can be fatal<br />
<strong>for</strong> rambutan trees if not controlled (MORTON, 1987a).<br />
6.1.3 Economics in Sri Lanka<br />
There is not much in<strong>for</strong>mation available about <strong>the</strong> economics of rambutan in Sri Lanka.<br />
However, rambutan is grown on medium scale commercial cultivation in Sri Lanka and <strong>the</strong><br />
fruit has a high local demand (DOASL, 2007 and BARRY, 2007). The only data which could<br />
be found about <strong>the</strong> cultivation of rambutan are within <strong>the</strong> years 1995 to 1999 and show<br />
significant growth of <strong>the</strong> cultivation of rambutan with a production of 7168 metric tons and<br />
total land extent of 896 ha in <strong>the</strong> year 1999 (DOASL, 2007).<br />
Rambutan is a crop best suited to <strong>the</strong> mid-country and low-country wet zone of Sri Lanka.<br />
The main harvest season is July/August and sometimes, depending on <strong>the</strong> wea<strong>the</strong>r conditions,<br />
a small off-season harvest is ga<strong>the</strong>red during December to February.<br />
The crop has good export potential and is presently exported in very limited amounts to <strong>the</strong><br />
Gulf Region, France, Germany and UK. The most important variety is `Malwana Special´<br />
followed by `Malaysian Red´ and ’Malaysian Yellow´ and some o<strong>the</strong>r local cultivars.<br />
Presently <strong>the</strong> most important areas where rambutan is grown successfully are within <strong>the</strong><br />
districts of Gampaha and Colombo (DOASL, 2007).<br />
6.2 Annona (Annona <strong>sp</strong>.)<br />
Annona belongs to <strong>the</strong> family Annonaceae which number of genera and <strong>sp</strong>ecies is still<br />
debated. Thereby varies <strong>the</strong> number of genera from 40 to 119 and <strong>the</strong> number of <strong>sp</strong>ecies from<br />
500 to over 2000 (DE Q. PINTO et al., 2005, BAERTELS, 1990 and MAHDEEM, 1994).<br />
13
However, <strong>the</strong> most important genus is Annona which has a multitude of <strong>sp</strong>ecies. Most of<br />
<strong>the</strong>se <strong>sp</strong>ecies are originated to tropical America and only a few are native to tropical Africa.<br />
This report deals with four <strong>sp</strong>ecies which are selected by ICUC (2002) as relevant<br />
underutilised plants: Annona cherimola, A. muricata, A. squamosa and A. reticulata. Only A.<br />
muricata and A. squamosa were tested in <strong>the</strong> experiments.<br />
A. cherimola, A. muricata and A. squamosa have great commercial importance whereas A.<br />
reticulata is significant a local used crop. They all belong to <strong>the</strong> areas of tropical South<br />
America although <strong>the</strong>y can be found in most tropical and some subtropical regions around <strong>the</strong><br />
world. E<strong>sp</strong>ecially A. cherimola which can be cultivated in cooler and drier climates is widely<br />
<strong>sp</strong>read in subtropical areas. According to DE Q. PINTO et al. (2005) have A. muricata and A.<br />
squamosa <strong>the</strong> widest distribution.<br />
Annona trees are multipurpose trees. Not only <strong>the</strong> fruit is consumed widely but <strong>the</strong> tree is also<br />
a source <strong>for</strong> medical and industrial products. The most important processed products are juice,<br />
jam and ice-cream (ICUC, 2002 and PRASADA RAO, AZEEMODDIN and THIRUMALA RAO,<br />
1984).<br />
Seed oil can be won out of all <strong>sp</strong>ecies and contains a large number of chemical compounds,<br />
like flavonoids, alkaloids and acetogenins. There<strong>for</strong>e <strong>the</strong> oil is used <strong>for</strong> treatments of<br />
medicinal conditions, <strong>for</strong> example skin diseases, intentional worms, inflammation of <strong>the</strong> eye<br />
and anti HIV and cancer effects.<br />
6.2.1 Description of <strong>the</strong> plant/fruit<br />
Annona trees are mostly shrubs or small trees with an erect or <strong>sp</strong>reading tree crown and a<br />
grey-brownish, rough and corrugated bark. Most Annona <strong>sp</strong>ecies are deciduous trees,<br />
e<strong>sp</strong>ecially when cultivated in areas with a dry season and without irrigation. The size, shape,<br />
colour and taste of <strong>the</strong> fruits are very diverse between <strong>the</strong> single <strong>sp</strong>ecies.<br />
6.2.1.1 Annona cherimola<br />
According to MORTON (1987b) <strong>the</strong> fruit of Annona cherimola is <strong>the</strong> most appreciated fruit of<br />
<strong>the</strong> genus Annona. The common name is cherimoya but <strong>the</strong> name is often misapplied to <strong>the</strong><br />
custard apple (Annona reticulate) and to atemoya, which is a hyprid of A. cherimola and A.<br />
squamosa.<br />
The origin of <strong>the</strong> fruit is <strong>the</strong> tropical highland of Peru, Ecuador, Columbia and Bolivia where<br />
it was cultivated as an ancient crop by <strong>the</strong> Incas. Between 1400 and 2000 m above sea level<br />
with a temperature between 17 °C and 20 °C are <strong>the</strong> regions where cherimoya naturally<br />
14
grows. Nowadays cherimoya is grown in most countries of <strong>the</strong> tropical highland and <strong>the</strong><br />
subtropics, like Spain, Egypt and Italy. In 1880 <strong>the</strong> fruit was introduced to Sri Lanka (Ceylon)<br />
where it is cultivated at small scale (MORTON, 1987b).<br />
The cultivation of cherimoya has commercial importance in Chile, Bolivia, Spain, United<br />
States (Florida) and New Zealand (GEORGE and NISSEN, 1991).<br />
The deciduous cherimoya tree is small, erect and somewhat <strong>sp</strong>reading and reaches a height of<br />
5 to 9 m. It has a shrub-like appearance, because of <strong>the</strong> in ground level frequently divided<br />
stem.<br />
The alternate leaves are 2 to 4 ranked and ovate-lanceolate to elliptical in shape with tiny and<br />
hairy petioles. The leaves are 10 to 25 cm long, 4 to 9 cm wide, slightly hairy on <strong>the</strong> upper<br />
surface and brownish velvety-tomentose on <strong>the</strong> underside. The flowers have a good fragrance<br />
and grow extra-axillary mostly solitary and opposite a leaf at <strong>the</strong> base of a branchlet.<br />
The flowers have a white-pinkish colour and are quite small. The<br />
shape of <strong>the</strong> fruit is normally conical, oval or somewhat heart<br />
shaped (Figure 6). But <strong>the</strong> <strong>for</strong>m is very varied due to irregular<br />
pollination. The fruit is 10 to 15 cm long, 5 to 10 cm wide and<br />
has an average weight of 150 to 700 g. In most varieties <strong>the</strong><br />
surface is covered with small conical protuberances over <strong>the</strong><br />
carpel. The skin of <strong>the</strong> fruit is thin and has a greenish-yellow<br />
colour when fully ripe. The pulp is snow white, juicy and contains<br />
numerous hard, brown, 1.2 to 2 cm long and glossy seeds. The<br />
Figure 6: Annona<br />
cherimola fruit has a pleasing aroma and delicious, sub acid and fragrant<br />
flavour, like a mix of pineapple and banana (MORTON, 1987b and<br />
DE Q. PINTO et al., 2005). The male and female parts of <strong>the</strong> flower do not mature<br />
simultaneously. That is <strong>the</strong> reason of <strong>the</strong> inadequate natural pollination of cherimoya. To<br />
reach a high fruit set rate <strong>the</strong> farmers are <strong>for</strong>ced to do hand pollination (MORTON, 1987b).<br />
15
6.2.1.2 Annona muricata<br />
Annona muricata is <strong>the</strong> <strong>sp</strong>ecies out of <strong>the</strong> genus Annona which produces <strong>the</strong> biggest flowers<br />
as well as <strong>the</strong> biggest fruits. The common English name is soursop and <strong>the</strong> Sri Lankan call <strong>the</strong><br />
plant “katu anoda”. There is no clarity about <strong>the</strong> exact origin of <strong>the</strong> fruit. Most authors opine<br />
that soursop is native to Central America, <strong>the</strong> Antilles or <strong>the</strong><br />
nor<strong>the</strong>rn South America (DE Q. PINTO et al., 2005, MORTON,<br />
1987b and MAHDEEM, 1994). The soursop was one of <strong>the</strong> first<br />
fruits carried out from South America to <strong>the</strong> Old World<br />
tropics where it has become widely distributed, from<br />
sou<strong>the</strong>aster China to Australia and <strong>the</strong> warm lowlands of<br />
eastern and western Africa. It is also found in Sri Lanka up to<br />
en elevation of 460 m above sea level (DE Q. PINTO et al.,<br />
2005).<br />
The soursop tree has a height of 4 to 10 m and is described as<br />
a low branching and bushy but slender tree (Figure 7). The<br />
evergreen tree has alternated, smooth and glossy leaves with<br />
short petioles (3 to 7 mm long). The upper surface is dark<br />
green whereas lighter beneath. The leaves are oblong-ovate to<br />
cylindrical, 6 to 20 cm long and 3 to 7 cm wide.<br />
Figure 7: Annona muricata<br />
tree<br />
The plant has very big flowers compared to <strong>the</strong> o<strong>the</strong>r Annona <strong>sp</strong>ecies, being 3 to 5 cm long.<br />
The greenish-yellow flowers born single and emerge anywhere on <strong>the</strong> trunk, branches or<br />
twigs.<br />
Figure 8: Annona<br />
muricata<br />
The fruits of Annona muricata are oval or heart-shaped,<br />
sometimes irregular due to bad pollination (Figure 8). The size of<br />
<strong>the</strong> fruit is very variable in a range from 10 to 30 cm in length and<br />
up to 20 cm in width with a weight of 0.5 to 10 kg. The skin of <strong>the</strong><br />
fruit has many short, fleshy and pointed protuberances and is<br />
popularly regarded as “<strong>sp</strong>iny”. The colour is dark green when<br />
unripe and turns into a slighter yellowish-green when ripe. Its<br />
inner surface is cream-coloured and granular and separates easily<br />
from <strong>the</strong> mass of snow-white, cotton-fibrous, juicy segments<br />
surrounding <strong>the</strong> central, soft-pithy core.<br />
16
MORTON (1987b) describes <strong>the</strong> aroma of <strong>the</strong> fruit as follows, <strong>the</strong> pulp is somewhat pineapple<br />
like, but its musky, sub acid to acid flavour is unique.<br />
The fruits have a large number of oval, smooth, hard and black seeds, up to 170 in large fruits.<br />
6.2.1.3 Annona squamosa<br />
Sugar apple or sweetsop are common English names <strong>for</strong> Annona squamosa. The sugar apple<br />
has <strong>the</strong> Sri Lankan name “weli anoda” and is <strong>the</strong> most widely grown of all Annona <strong>sp</strong>ecies.<br />
The original home of <strong>the</strong> fruit is not really known. According to MAHDEEM (1994) Annona<br />
squamosa seems to be native in south eastern Mexico. MORTON (1987b) is of <strong>the</strong> opinion that<br />
<strong>the</strong> origin is unknown. DE Q. PINTO et al. (2005) explains that <strong>the</strong> plant is native to circum<br />
Caribbean or <strong>the</strong> nor<strong>the</strong>rn South America lowlands. Today <strong>the</strong> tree can be found in nearly all<br />
tropical countries.<br />
Annona squamosa requires tropical or near tropical climates and has a high drought tolerance.<br />
It grows best in dry areas. In Sri Lanka <strong>the</strong> sugar apple flourishes in <strong>the</strong> wet as well as in <strong>the</strong><br />
dry zones from sea level to 1100 m elevation (MORTON, 1987b).<br />
The deciduous tree reaches a height of 3 to 6 m with an open crown of lateral branches. The<br />
alternated leaves are lanceolate or oblong, blunt tipped, 5 to 17 cm long and 2 to 7 cm wide,<br />
with an obtuse apex. The colour is brilliant-green on <strong>the</strong> upper site and bluish-green with a<br />
bloom below.<br />
The flowers of <strong>the</strong> sugar apple trees are similar in size and <strong>for</strong>m to <strong>the</strong> ones of <strong>the</strong> cherimoya<br />
tree.<br />
The fruit is globose, heart-shaped or conical in <strong>for</strong>m with a diameter of 5 to 10 cm and a<br />
weight of 120 to 330 g (Figure 9). According to MORTON (1987b) <strong>the</strong> surface of <strong>the</strong> fruit,<br />
which is covered with tuberculates and a powdery bloom resembles a “hand-grenade.” The<br />
tuberculates are separating when <strong>the</strong> fruit is ripe so that <strong>the</strong> fruit falls to pieces.<br />
The creamy white and custard like pulp has a delightfully fragrant and a pleasant sweet-sour<br />
flavour. The fruit contains 35 to 45 seeds which are oblong-cylindrical, dark brown and about<br />
1.5 to 2 cm long.<br />
Pollination and fruit set problems are similar to those of o<strong>the</strong>r annonas.<br />
Annona squamosa has a few cultivars including some varieties with a red coloured skin, like<br />
A. squamosa `Red´, A. squamosa `Red-<strong>sp</strong>eckled´ and A. squamosa `Crimson´ (Figure 10).<br />
17
Figure 9: Annona squamosa<br />
Figure 10: A red skinned variety<br />
of Annona squamosa<br />
6.2.1.4 Annona reticulata<br />
DE Q. PINTO et al. (2005) considers that Annona reticulata is <strong>the</strong> most vigorous of <strong>the</strong> annona<br />
<strong>sp</strong>ecies described in this report. MORTON (1987b) however, reports that both in tree and in<br />
fruit <strong>the</strong> <strong>sp</strong>ecies is generally rated as <strong>the</strong> mediocre or “ugly duckling” <strong>sp</strong>ecies among <strong>the</strong><br />
prominent members of <strong>the</strong> genus Annona.<br />
The common English name of Annona reticulata is bullock’s-heart or custard apple.<br />
In <strong>the</strong> past many people thought that <strong>the</strong> origin of <strong>the</strong> custard apple is West India but today it<br />
is proven that <strong>the</strong> plant is native to Central or nor<strong>the</strong>rn South America (MORTON, 1987b, DE<br />
Q. PINTO et al., 2005 and MAHDEEM, 1994). Today <strong>the</strong> tree is<br />
found like <strong>the</strong> o<strong>the</strong>rs a<strong>for</strong>e described, in many tropical countries<br />
around <strong>the</strong> world.<br />
Annona reticulata grows well in tropical climates with cool<br />
winter times and prefers a humid atmo<strong>sp</strong>here. The tree is<br />
tolerant to cold temperatures. According to MORTON (1987b) a<br />
fully grown custard apple tree survives temperatures of 2.8 °C<br />
without serious harm.<br />
Figure 11: Annona<br />
reticulata<br />
The deciduous Annona reticulata tree is small and tender with an open and irregular crown<br />
which can reach a height of 5 to 10 m. The alternate leaves are oblong or narrow-lanceolate,<br />
dark-green and glabrous with a length of 10 to 25 cm and a width of 2 to 7 cm.<br />
The flowers are similar in <strong>for</strong>m to those of Annona squamosa and Annona cherimola. The<br />
only different is that <strong>the</strong>y are generally grown in drooping clusters with 2 to 10 flowers. The<br />
0.1 to 1 kg heavy fruit is commonly heart-shaped but sometimes different like oval, lopsided<br />
or nearly round (Figure 11). They are 8 to 15 cm in diameter, coriaceous and have a reddishyellow<br />
surface colour with impressed lines above <strong>the</strong> carpel.<br />
18
The flesh is creamy-white with a juicy aromatic to hard flavour with a repulsive taste<br />
according to <strong>the</strong> cultivar. According to DE Q. PINTO et al. (2005) <strong>the</strong> custard apple is <strong>the</strong> least<br />
tasty fruit of <strong>the</strong> cultivated annona <strong>sp</strong>ecies.<br />
The seeds which are dark-brown or black in colour, oblong, smooth and less <strong>the</strong>n 1.25 cm<br />
long are commonly more than 40 in number.<br />
6.2.2 Main diseases<br />
According to DE Q. PINTO et al. (2005) are Annona trees attacked by a large number of insect<br />
pests and numerous diseases, although many of <strong>the</strong>m are not economically important. The<br />
most important pests are aphids, mealy- and scale-bugs and fruit-flies (FRANKE, 1994).<br />
According to DE Q. PINTO et al. (2005) <strong>the</strong> annona moth (Cerconota annonella), commonly<br />
known as <strong>the</strong> “fruit borer” is <strong>the</strong> most important of <strong>the</strong> insect pests attacking Annona <strong>sp</strong>ecies.<br />
The most important root diseases caused by fungi are damping-off (Rhizoctonia solani and<br />
Fusarium <strong>sp</strong>p.) and black root rot (Phytophthora <strong>sp</strong>p., Cylindrocladium clavatum and<br />
Sclerotium rolfsii) (DE Q. PINTO et al., 2005). The bacterial wilt is also an important basal and<br />
root rot which affects mainly seedlings in nurseries and grafted trees in <strong>the</strong> field (PLOETZ,<br />
2003). There are several diseases attacking <strong>the</strong> fruits of Annona <strong>sp</strong>ecies during pre and post<br />
harvest phases: anthracnose (Glomerella cingulata), black cancer (Phomopsis<br />
annonacearum), diplodia rot (<strong>Botryodiplodia</strong> <strong>the</strong>obromae), purple blotch (Phytophthora<br />
palmivora) and brown rot (Rhizopus stolonifer) (DE Q. PINTO et al., 2005 and PLOETZ, 2003).<br />
6.2.3 Economics in Sri Lanka<br />
There is only little in<strong>for</strong>mation available about <strong>the</strong> economic situation of annona <strong>sp</strong>ecies in Sri<br />
Lanka. In <strong>the</strong> course of his stay in Sri Lanka <strong>the</strong> reports author could not find any annona<br />
plantations. It seems that Annona trees are mostly planted in home gardens. The same<br />
situation could be seen on <strong>the</strong> markets where Annona <strong>sp</strong>ecies, e<strong>sp</strong>ecially soursop and sugar<br />
apple, were available but not in high quantities. By means of some market sellers cherimoya<br />
is imported to Sri Lanka in small quantities.<br />
7 The disease (<strong>Botryodiplodia</strong> <strong>sp</strong>.)<br />
7.1 Taxonomy<br />
<strong>Botryodiplodia</strong> <strong>sp</strong>. belong to <strong>the</strong> class of Ascomycetes and live in a saprophytic way. They are<br />
moulds which normally need injured tissue to parasite <strong>the</strong> plant.<br />
19
Characteristically <strong>for</strong> this fungus is <strong>the</strong> generation of pycnidia in which <strong>the</strong> <strong>sp</strong>ores of <strong>the</strong><br />
fungus are <strong>for</strong>med. Pycnidia develop on artificial media only rarely and after a long time. But<br />
if <strong>the</strong>y are generated <strong>the</strong>y can be seen with <strong>the</strong> naked eye as black balls of <strong>the</strong> size of a<br />
pinhead.<br />
The <strong>sp</strong>ores are elliptical and compared to o<strong>the</strong>r <strong>sp</strong>ores relatively big. Young, immature <strong>sp</strong>ores<br />
are colourless and unicellular whereas mature <strong>sp</strong>ores are brown coloured, distichous and<br />
thick-walled (Figure 12). Only <strong>the</strong> presence of <strong>the</strong> mature <strong>sp</strong>ores allows a proper<br />
identification of <strong>the</strong> fungus.<br />
The fast and constantly growing mycelium of <strong>the</strong> fungus is snow-white at first, turning its<br />
colour within three to four weeks black (Figure 13). This discolouring is not due to <strong>the</strong><br />
generating of <strong>the</strong> <strong>sp</strong>ores.<br />
The most important <strong>sp</strong>ecies of <strong>the</strong> genus is <strong>Botryodiplodia</strong> <strong>the</strong>obromae.<br />
Figure 12: Mature and immature <strong>sp</strong>ores of <strong>Botryodiplodia</strong> <strong>sp</strong>.<br />
Figure 13: Petri-dishes with <strong>Botryodiplodia</strong> <strong>sp</strong>. after seven days (left)<br />
and after four weeks (right)<br />
20
7.2 Disease pattern<br />
<strong>Botryodiplodia</strong> <strong>sp</strong>. is a fungal disease attacking <strong>the</strong><br />
fruits of Annona <strong>sp</strong>ecies e<strong>sp</strong>ecially in neglected<br />
orchards. Diseased fruits show symptoms of<br />
purplish to black <strong>sp</strong>ots or blotches confined to <strong>the</strong><br />
surface of <strong>the</strong> fruit and eventually covered with<br />
white mycelia and black pycnidia (Figure 14).<br />
Diplodia rot is distinguished by its dark internal<br />
discolouration and <strong>the</strong> extensive corky rotting<br />
produces (DE Q. PINTO et al., 2005). The<br />
penetrated flesh eventually softens or hardens and<br />
cracks, depending on <strong>the</strong> presence of secondary<br />
microbes.<br />
Figure 14: A.muricata fruit infected with<br />
diplodia rot<br />
8 Bio-control agent (Trichoderma <strong>sp</strong>.)<br />
Trichoderma <strong>sp</strong>. are fungi that are present in nearly all soils and o<strong>the</strong>r diverse habitats such as<br />
decaying wood. According to HARMANN (2005) <strong>the</strong> fungi are <strong>the</strong> most prevalent fungi in soil.<br />
They are favoured by <strong>the</strong> presence of high levels of plant roots, which <strong>the</strong>y colonize readily.<br />
In addition to that Trichoderma <strong>sp</strong>p. are able to attack, parasitize and o<strong>the</strong>rwise gain nutrition<br />
from o<strong>the</strong>r fungi. These abilities turn <strong>the</strong> fungi into a reputable bio-control agent.<br />
8.1 Taxonomy<br />
The sexual life-<strong>for</strong>m of <strong>the</strong> fungi of <strong>the</strong> genus Trichoderma is extensively unknown. So <strong>the</strong><br />
classification of <strong>the</strong> fungus to <strong>the</strong> class of Deuteromycetes results from <strong>the</strong> asexual life-<strong>for</strong>m.<br />
Reproduction of <strong>the</strong> ubiquitous genus is by conidia which are produced from highly branched<br />
conidiophores. These are arranged in irregular verticils, with <strong>the</strong> sub-terminal phialides borne<br />
more or less at right angles to <strong>the</strong> stipule. The shape of <strong>the</strong> phialides is typical <strong>for</strong><br />
Trichoderma <strong>sp</strong>. and is <strong>the</strong> crucial factor <strong>for</strong> <strong>the</strong> identification of <strong>the</strong> fungi (PITT and<br />
HOCKING, 1985) (Figure 15).<br />
21
Figure 15: Phialides of Trichoderma <strong>sp</strong>.<br />
Figure 16: Petri-dish with a six day<br />
old Trichoderma <strong>sp</strong>. colony<br />
Trichoderma <strong>sp</strong>. colonies <strong>sp</strong>read rapidly and have a loose textured mycelium which<br />
characteristically develops irregularly, with tufts or isolated patches. The mycelium has a<br />
white tran<strong>sp</strong>arent colour. The small conidia are in general green coloured and smooth and<br />
develop after a short time of incubation. That is <strong>the</strong> reason why <strong>the</strong> mycelium is turning into<br />
green after a few days (Figure 16).<br />
8.2 The role of mycoparasitism<br />
As a<strong>for</strong>ementioned Trichoderma <strong>sp</strong>p. are already used as<br />
bio-control agents against phytopathogenic fungi and<br />
bacteria. Pythium, Phytophthora, Rhizoctonia, Fusarium<br />
and Verticillium are fungi against which Trichoderma <strong>sp</strong>.<br />
have already made an impact (KORTING, 2001). The<br />
reasons <strong>for</strong> this use are certain antagonistic attributes<br />
which most fungi out of <strong>the</strong> genus Trichoderma own. The<br />
most important mechanisms are, according to WANTOCH-<br />
REKOWSKI (2004), mycoparasitism, antibiosis and competition.<br />
Trichoderma <strong>sp</strong>. are able to enter into <strong>the</strong> mycelium<br />
of o<strong>the</strong>r fungi. There<strong>for</strong>e <strong>the</strong> fungi wind around <strong>the</strong><br />
host’s mycelium and build hyphae which break into <strong>the</strong><br />
host’s mycelium (Figure 17). In this way <strong>the</strong> antagonists<br />
are able to “suck” <strong>the</strong> hosts.<br />
Figure 17: Trichoderma<br />
harisianum parasitizes Pythium<br />
ultimum from (BENHAMOU and<br />
CHET, 1997 and WANTOCH-<br />
REKOWSKI, 2004)<br />
22
The fungi are producing enzymes such as cellulases and chitinases which are able to destroy<br />
<strong>the</strong> cell walls of o<strong>the</strong>r fungi or inhibit <strong>the</strong> germination of o<strong>the</strong>r <strong>sp</strong>ores. Some Trichoderma<br />
strains compose antibiotics which can control bacterial phytopathogenics). These mechanisms<br />
are called antibiosis.<br />
According to WILKE (2001) Trichoderma provides <strong>the</strong> same compounds as plant <strong>for</strong>tifier<br />
because it produces hormones and enzymes which are supporting <strong>the</strong> growth of <strong>the</strong> cultural<br />
plant. Apart from parasitism and antibiosis Trichoderma <strong>sp</strong>. can compete with o<strong>the</strong>r fungi<br />
against nutrients and <strong>sp</strong>ace.<br />
9 Experiments<br />
9.1 Experimental procedure<br />
The first step of <strong>the</strong> experiment which was undertaken by <strong>the</strong> author was to collect soil<br />
samples from different regions of Sri Lanka. The samples were taken from rambutan<br />
plantations and accordingly from soils straight underneath Annona trees. Afterwards<br />
Trichoderma <strong>sp</strong>p. were isolated out of <strong>the</strong> soil samples.<br />
In a second step bio-assays were carried out to prove <strong>the</strong> effectiveness of <strong>the</strong> Trichoderma <strong>sp</strong>.<br />
against <strong>the</strong> pathogens, which has been isolated earlier from infected fruits.<br />
In ano<strong>the</strong>r experiment, cross-inoculations were done to analyse whe<strong>the</strong>r diseased fruits of<br />
various <strong>sp</strong>ecies infect each o<strong>the</strong>r.<br />
Fur<strong>the</strong>rmore a <strong>sp</strong>orulation experiment was undertaken.<br />
9.1.1 Laboratory set-up<br />
The most important thing while working in microbiological laboratories is to work under<br />
sterile conditions. There<strong>for</strong>e <strong>the</strong> working places and all materials, as well as <strong>the</strong> tools and<br />
some chemicals have to be sterilized.<br />
The sterilization of most of <strong>the</strong> glassware, such as petri-dishes was done in a sterilization<br />
oven. The mode of action of <strong>the</strong> oven is hot dry air sterilization, in which at 180 °C in one<br />
hour’s time most organisms are killed. Hot moist air under pressure is <strong>the</strong> mode of action of<br />
<strong>the</strong> autoclaves (Figure 18). They are used <strong>for</strong> liquid sterilizations like media and water and<br />
o<strong>the</strong>r utilities such as funnels, beakers and pipettes as well as <strong>for</strong> things made out of<br />
polycarbonates like <strong>the</strong> pipette-tips. Autoclaves work at a temperature of 121 °C and a<br />
pressure of 1.1 kg/cm² and need only 15 minutes <strong>for</strong> sterilization. The reason why <strong>the</strong><br />
23
autoclave is used <strong>for</strong> liquid sterilization is due to <strong>the</strong> lower temperature. Many chemicals are<br />
degenerating at a temperature more than 121 °C.<br />
Figure 18: Autoclave at ITI<br />
Figure 19: Laminar flow cabinet at ITI<br />
For <strong>the</strong> sterilization of <strong>the</strong> surface of <strong>the</strong> working place and <strong>the</strong> workers hands a 70 % ethanol<br />
solution is used. It is also used <strong>for</strong> <strong>the</strong> working utensils such as scalpel and <strong>for</strong>ceps while<br />
working by dipping <strong>the</strong>m into <strong>the</strong> alcohol solution and flaming <strong>the</strong>m afterwards in <strong>the</strong> Bunsen<br />
burner flame.<br />
To sterilize <strong>the</strong> laminar flow cabinet or <strong>the</strong> incubator a so called fumigation should be done by<br />
filling a little amount of a 8 % <strong>for</strong>maldehyde dilution into a beaker or Petri-dish and keep it<br />
<strong>for</strong> more than a hour, when possible over night, in <strong>the</strong> closed incubator or laminar of flow<br />
cabinet.<br />
In all experiments which are described in this report <strong>the</strong> work was done under sterile<br />
conditions by using <strong>the</strong> above mentioned methods.<br />
The work with micro-organisms always takes place in so called laminar flow cabinet (Figure<br />
19). This is a half closed or manual closable glass cabin in which a constant air flow takes<br />
place and <strong>the</strong> air gets filtered to avoid contaminations.<br />
The medium which was used <strong>for</strong> all workings and experiments was a Potato-Dextrose-Agar<br />
(PDA) medium. The medium was prepared fresh, immediately be<strong>for</strong>e every single trial series.<br />
24
250 ml PDA was made up as follows:<br />
Material:<br />
• Bunsen burner<br />
• Screw cap bottle (300 ml)<br />
• Measuring cylinder (500 ml)<br />
• Funnel<br />
• Cloth <strong>for</strong> filtering<br />
• Beaker (500 ml)<br />
• Heating plate<br />
Chemicals:<br />
• 50 g potatoes<br />
• 5 g glucose<br />
• 5 g agar<br />
After washing, removing <strong>the</strong> skin and cutting into little quarters <strong>the</strong> potatoes have to boil in<br />
250 ml distilled water <strong>for</strong> 20 minutes. After boiling <strong>the</strong> solution has to be filtered through a<br />
funnel using a layer of muslin made up to a volume of 250 ml with distilled water. The<br />
solution is decanted into <strong>the</strong> screw cap bottle and <strong>the</strong> glucose and <strong>the</strong> sugar are added. After<br />
autoclaving <strong>the</strong> pH of <strong>the</strong> medium can be adjusted by adding 1.6 ml sterile tartaric acid per<br />
100 ml PDA to avoid bacterial growth. Lastly, <strong>the</strong> PDA is poured into sterile petri dishes<br />
(plates). In doing so <strong>the</strong> neck of <strong>the</strong> PDA-filled bottle should be held into <strong>the</strong> Bunsen burner<br />
flame once in a while to avoid contaminations. 250 ml<br />
PDA should be enough <strong>for</strong> 15 plates.<br />
During <strong>the</strong> experiments <strong>the</strong> cultures where stored in<br />
incubators which were set up to a temperature of 28 °C<br />
(Figure 20). The Trichoderma <strong>sp</strong>. cultures were put into an<br />
extra incubator. The reason <strong>for</strong> this is that Trichoderma <strong>sp</strong>.<br />
contaminate o<strong>the</strong>r cultures due to <strong>the</strong> very small and<br />
numerous conidia very easily.<br />
9.1.2 Soil sampling<br />
Figure 20: Incubator at ITI<br />
Soil samples from under rambutan and annona trees were taken in various regions of Sri<br />
Lanka. The samples were taken within <strong>the</strong> soil area which was covered by <strong>the</strong> crown of <strong>the</strong><br />
tree. Out of this area four samples were taken randomly (Figure 21). Only <strong>the</strong> top soil to a<br />
deepness of 2 cm was taken and filled into plastic bags which were labelled and closed with a<br />
loose burl to guarantee an air exchange.<br />
25
Figure 21: Soil sampling in a rambutan plantation<br />
In this manner 16 soil samples were taken from 4 different rambutan plantations and 13<br />
samples from annona trees from 3 different areas. 7 of <strong>the</strong> “annona samples” were taken from<br />
Annona squamosa trees and 6 from Annona muricata trees; all <strong>the</strong> trees were standing in<br />
private gardens or little home gardens.<br />
Table 1 gives an overview of <strong>the</strong> soil samples and <strong>the</strong>ir place of sourcing.<br />
Table 1: An Overview of <strong>the</strong> soil samples which were taken<br />
District<br />
City<br />
Gampaha District Urapola<br />
Quantity soil samples of:<br />
rambutan A. squamosa A. muricata<br />
1 st plantation 4<br />
2 nd plantation 4<br />
3 rd plantation 4<br />
Date<br />
3. May<br />
Kegalle District Warakapola 4 8. June<br />
Anuradhapura Wijayapura 2 1 10. May<br />
District Medawachchiya 3 11. May<br />
Kegalle District Galagedera 1 12. May<br />
Matale District Matale 2 4 26. May<br />
9.1.3 Isolation of <strong>the</strong> bio-control agent<br />
To find <strong>the</strong> bio-control agent in <strong>the</strong> soil samples you have to see total number of fungi which<br />
are present in <strong>the</strong> soil. If Trichoderma <strong>sp</strong>. is present within <strong>the</strong> fungi an isolation can be done.<br />
26
The following materials and chemicals are needed to analyse a soil sample regarding <strong>the</strong><br />
population of <strong>the</strong> fungi:<br />
Materials:<br />
• Bunsen burner<br />
• 2 flasks<br />
• Funnel carped with cotton wool<br />
• 6 closable tubes<br />
• Micropipette <strong>for</strong> 1 ml with 20<br />
pipette-tips<br />
• Petri-dishes filled with PDA<br />
Chemicals:<br />
• 10 g soil<br />
• 150 ml distilled water<br />
To analyse a soil sample a <strong>sp</strong>ore su<strong>sp</strong>ension has to be done be<strong>for</strong>e. There<strong>for</strong>e 10 g of <strong>the</strong> soil<br />
sample was placed in a flask to which 10 ml distilled water was added and shaken carefully in<br />
a circular movement <strong>for</strong> 20 minutes. The solution was <strong>the</strong>n filtered through a funnel with<br />
cotton wool into a second flask and in doing so <strong>the</strong> <strong>sp</strong>ore solution was obtained.<br />
Normally <strong>the</strong> <strong>sp</strong>ore concentration of <strong>the</strong> <strong>sp</strong>ore solution is too high and a dilution series has to<br />
be done. There<strong>for</strong>e 1 ml of <strong>the</strong> <strong>sp</strong>ore solution was used with 9 ml distilled water to obtain a<br />
10 -1 dilution. This dilution procedure was repeated until a dilution series with <strong>the</strong> powers 10 -1<br />
to 10 -6 was obtained.<br />
0.1 ml of each dilution was <strong>sp</strong>read on petridishes<br />
filled with PDA and incubated. These<br />
cultures (Figure 22) were observed every day <strong>for</strong><br />
one week to check wea<strong>the</strong>r Trichoderma <strong>sp</strong>. is<br />
appearing somewhere.<br />
If <strong>the</strong> existence of <strong>the</strong> wanted fungus<br />
(Trichoderma <strong>sp</strong>.) was <strong>sp</strong>eculated an in<strong>sp</strong>ection<br />
by using <strong>the</strong> microscope was undertaken. If <strong>the</strong><br />
identification was positive an isolation of <strong>the</strong><br />
fungus to a pure culture was undertaken.<br />
Figure 22: Seven days old plate from<br />
a soil experiment<br />
27
9.1.4 Isolation of <strong>the</strong> pathogen<br />
Materials:<br />
• Bunsen burner<br />
• 4 beakers<br />
• Scalpel and <strong>for</strong>ceps<br />
• 1 empty petri-dish<br />
• Measuring cylinder (10 ml)<br />
• Pure PDA plates<br />
Chemicals:<br />
• 100 ml ethanol solution (70 %)<br />
• Sodium hypochlorite solution (5 %)<br />
• 250 ml distilled water<br />
For <strong>the</strong> isolation of <strong>the</strong> pathogen partially diseased fruit are required. The fruit should have<br />
enough healthy tissue. The fruit which were <strong>the</strong>re<strong>for</strong>e bought ei<strong>the</strong>r from local markets or<br />
collected during <strong>the</strong> field trips.<br />
From <strong>the</strong> fruit tissue squares were taken with <strong>the</strong> size of 1 cm² and <strong>the</strong> thickness of 3 mm. It<br />
was important that only 1/3 of <strong>the</strong> tissue was diseased. The tools which were necessary <strong>for</strong><br />
this had to be sterilized after every cut (scalpel and <strong>for</strong>ceps). The cuttings took place in an<br />
empty petri-dish.<br />
The pieces obtained were surface sterilized by dipping <strong>the</strong>m<br />
<strong>for</strong> three minutes in a 5 % sodium hypochlorite solution. The<br />
solution was prepared immediately be<strong>for</strong>e <strong>the</strong> surface<br />
sterilization, because <strong>the</strong> effectiveness of <strong>the</strong> solution<br />
decelerates with running time.<br />
After that <strong>the</strong> pieces were washed twice by dipping <strong>the</strong>m into<br />
Figure 23: Isoloation of <strong>the</strong> distilled water <strong>for</strong> three minutes to remove <strong>the</strong> rests of <strong>the</strong><br />
pathogen on rambutan (five<br />
sterilization solution. In each case two washed fruit pieces<br />
days old plate)<br />
were put into one PDA plate and incubated. The mycelium of<br />
<strong>the</strong> pathogen covered <strong>the</strong> whole plate after a few days (Figure 23) and could be subcultivated.<br />
The pathogenicity of <strong>the</strong> isolated organism was proved using Koch’s postulates. Koch’s<br />
postulates can be summarised in four steps (FREDRICKS and RELMAN, 1996):<br />
• The organism must be found in all plants suffering from <strong>the</strong> disease, but not in<br />
healthy plants.<br />
• The organism must be isolated from a diseased plant and grown in pure culture.<br />
• The cultured organism should cause disease when introduced into a healthy plant.<br />
• The organism must be reisolated from <strong>the</strong> experimentally infected plant.<br />
28
If <strong>the</strong> Koch’s postulates are fulfilled it is proven that <strong>the</strong> isolated pathogen and <strong>the</strong> pathogen<br />
which causes <strong>the</strong> disease of <strong>the</strong> fruit are identical.<br />
9.1.5 Bio-Assays<br />
Materials:<br />
• Bunsen burner<br />
• Cork borer (number 5)<br />
• Spatula<br />
• Micropipette and pipette-tips<br />
For <strong>the</strong> <strong>sp</strong>ore su<strong>sp</strong>ension:<br />
• Small flask<br />
• Loop<br />
• Measuring cylinder (10 ml)<br />
• Funnel with cotton wool<br />
Chemicals:<br />
• 150 ml Ethanol (70 %)<br />
• 50 ml distilled water<br />
The so called “bio-assays” are in vitro experiments and should give in<strong>for</strong>mation about <strong>the</strong><br />
activity of <strong>the</strong> bio-control agents (BC-agent) against <strong>the</strong> pathogens. There<strong>for</strong>e several<br />
experimental series were undertaken, which re<strong>sp</strong>ectively proved two BC-agents against four<br />
pathogens.<br />
The activity of <strong>the</strong> BC-agents was proved in petri-dishes filled with 15 ml PDA. Near <strong>the</strong><br />
outer border of <strong>the</strong> Petri-dish four wells has been cut into <strong>the</strong> medium with a constant distance<br />
(Figure 24). The wells were cut with a cork<br />
borer number 5 (1 cm in diameter). Afterwards<br />
0.8 ml of a <strong>sp</strong>ore su<strong>sp</strong>ension of a BCagent<br />
was filled in each well. Following this<br />
a mycelia disk of <strong>the</strong> pathogen, which was<br />
cut with <strong>the</strong> same cork borer (No. 5) from a<br />
7 days old culture, was placed in <strong>the</strong> middle Figure 24: Preparation of a petri-dish <strong>for</strong> a bioassay<br />
of <strong>the</strong> petri-dish (Figure 24).<br />
The <strong>sp</strong>ore su<strong>sp</strong>ension was won out of 5 to 10 days old cultures of <strong>the</strong> BC-agents according to<br />
<strong>the</strong> time of full <strong>sp</strong>orulation. There<strong>for</strong>e 20 mm distilled water was filled into <strong>the</strong> culture plate<br />
and <strong>the</strong> upper layer of <strong>the</strong> fungus was scraped off <strong>the</strong> medium. This mix of distilled water and<br />
fungus tissue was filtered to receive a pure <strong>sp</strong>ore su<strong>sp</strong>ension of <strong>the</strong> BC-agent. For <strong>the</strong> controls<br />
<strong>the</strong> wells were filled with distilled water. In this manner five replications per pathogen took<br />
place, so that one experimental series consisted out of 60 culture plates.<br />
29
The effectivity of <strong>the</strong> BC-agent was established by doing daily measurements which recorded<br />
<strong>the</strong> diameter of <strong>the</strong> pathogen between <strong>the</strong> wells. If <strong>the</strong> diameter of <strong>the</strong> pathogen was getting<br />
smaller during <strong>the</strong> experimental time or <strong>the</strong> pathogen disappeared completely, <strong>the</strong> BC-agent<br />
had a good activity. If <strong>the</strong> BC-agent had no activity against <strong>the</strong> pathogen <strong>the</strong> diameter of <strong>the</strong><br />
pathogen increased and <strong>the</strong> BC-agent could not develop.<br />
Each experimental series was incubated <strong>for</strong> 7 days at a temperature of 28 °C and daily<br />
observed (Figure 25 and Figure 26).<br />
Figure 25: BC-agent with a good (right) and a bad (left) effectiveness<br />
Figure 26: Experimental series of bio-assay after 2 (left) and 6 (right) days<br />
9.1.6 Cross-inoculation<br />
The cross-inoculation experiment is used as an examination if <strong>the</strong> pathogens which were<br />
isolated from <strong>the</strong> various fruits are host <strong>sp</strong>ecific or if <strong>the</strong>y are able to infect <strong>the</strong> o<strong>the</strong>r fruits<br />
with <strong>the</strong> same aggressiveness.<br />
30
The experiment was carried out on rambutan, sugar apple and soursop fruits and repeated.<br />
In every experimental trial four pathogens were tested on ten fruits and two different controls<br />
were undertaken, in which likewise ten fruits were used. So that in total 60 fruits were<br />
necessary <strong>for</strong> one experimental series. The soursops and sugar apples were bought from local<br />
markets in Colombo whereas <strong>the</strong> rambutan fruits were picked direct from a rambutan<br />
plantation. The experiments undertaken with rambutan were only done with fruits of <strong>the</strong><br />
variety `Malwana Special´.<br />
At <strong>the</strong> beginning of <strong>the</strong> experiments <strong>the</strong> fruits had to be surface sterilized to avoid field<br />
inoculations. There<strong>for</strong>e <strong>the</strong> fruits were dipped <strong>for</strong> five minutes into a 5 % sodium<br />
hypochlorite solution. After drying <strong>the</strong> fruits on air in <strong>the</strong> laminar flow cabinet one well was<br />
cut near <strong>the</strong> stem by using a cork borer (No. 5). It was important that <strong>the</strong> well was not cut too<br />
deep into <strong>the</strong> fruit at best only <strong>the</strong> skin should be removed. Than a seven day old mycelia disk<br />
of <strong>the</strong> pathogen was insert into <strong>the</strong> well.<br />
In <strong>the</strong> first control (control 1) <strong>the</strong> wells were filled with distilled water whereas <strong>the</strong> second<br />
control (control 2) consisted out of untreated fruits.<br />
All fruits where stored in self-made humidity chambers (Figure 27) during <strong>the</strong> experimental<br />
time of six days. An observation of <strong>the</strong> fruits was undertaken daily which ga<strong>the</strong>red <strong>the</strong> disease<br />
<strong>sp</strong>read in percent by using an infestation key (Figure 28 and Figure 29).<br />
Figure 27: Humidity chamber <strong>for</strong> <strong>the</strong> rambutan<br />
Cross-inoculation experiment<br />
Figure 28: Infestation key <strong>for</strong> crossinoculation<br />
experiment with sugar apple<br />
31
Figure 29: Experimental series of a cross-inoculation on Annona squamosa<br />
(day 2 to day 6)<br />
9.1.7 Sporulation experiment<br />
As mentioned in chapter 7 <strong>Botryodiplodia</strong> <strong>sp</strong>. under artificial conditions need a very long time<br />
to produce <strong>sp</strong>ores, which are crucial <strong>for</strong> <strong>the</strong> identification of <strong>the</strong> fungus. There<strong>for</strong>e two<br />
different methods were tested which possibly affect <strong>the</strong> <strong>sp</strong>orulation of <strong>Botryodiplodia</strong> <strong>sp</strong>. in<br />
consideration of its <strong>sp</strong>eed.<br />
The different <strong>Botryodiplodia</strong> <strong>sp</strong>. cultures were exposed to three different conditions.<br />
First all cultures were subcultured nine times and incubated <strong>for</strong> five days at 28 °C to obtain<br />
completely grown plates. Following <strong>the</strong> cultures were divided into three sets each with three<br />
plates.<br />
One set remained in <strong>the</strong> incubator, <strong>the</strong> second one was exposed to <strong>the</strong> daylight and <strong>the</strong> third<br />
one was exposed to daylight after a cold shock was admitted by storing <strong>the</strong> culture plates <strong>for</strong><br />
seven days at 13 °C in a cold room.<br />
The experimental time was one month while <strong>the</strong> cultures were analysed regarding to <strong>sp</strong>ores<br />
twice a week.<br />
9.2 Results<br />
Trichoderma <strong>sp</strong>. found in <strong>the</strong> soil samples:<br />
In total twelve Trichoderma <strong>sp</strong>. could be isolated from six sources, which were different<br />
concerning <strong>the</strong>ir morphology and <strong>the</strong>ir development (Figure 30). The exact differences<br />
between <strong>the</strong> several isolates are found in <strong>the</strong> Annexes. Table 2 gives an overview of <strong>the</strong><br />
isolated Trichoderma <strong>sp</strong>. regarding <strong>the</strong>ir source and <strong>the</strong> time <strong>the</strong>y need to full <strong>sp</strong>orulation of<br />
<strong>the</strong> plate.<br />
32
Table 2: An overview of <strong>the</strong> isolated Trichoderma <strong>sp</strong>.<br />
Code Source Fruit Full <strong>sp</strong>orulation<br />
Tra I Urapola after 5 days<br />
Tra II Warakapola Rambutan after 5 days<br />
Tra III Warakapola<br />
after 5 days<br />
Tmu I Galagedera after 10 days<br />
Tmu II Galagedera after 6 days<br />
Annona<br />
Tmu III Galagedera after 6 days<br />
muricata<br />
Tmu IV Matale after 6 days<br />
Tmu V Wijayapura<br />
after 5 days<br />
Tsq I Matale after 6 days<br />
Tsq II Matale Annona after 5 days<br />
Tsq III Wijayapura squamosa after 5 days<br />
Tsq IV Medawachchiya<br />
after 6 days<br />
Figure 30: The twelve Trichoderma<br />
<strong>sp</strong>. Found in <strong>the</strong> soil samples<br />
<strong>Botryodiplodia</strong> <strong>sp</strong>. isolated from <strong>the</strong> fruits:<br />
Eight <strong>Botryodiplodia</strong> <strong>sp</strong>. could be isolated out of different fruits. Thereby three<br />
<strong>Botryodiplodia</strong> <strong>sp</strong>. were isolated from rambutan, three from Annona squamosa and one from<br />
Annona muricata fruits. Moreover one fungus was isolated out of a red skinned Annona<br />
squamosa.<br />
The fungi isolated out of <strong>the</strong> same fruits did not differ from each o<strong>the</strong>r. So it was assumed that<br />
it is <strong>the</strong> matter of <strong>the</strong> same <strong>sp</strong>ecies.<br />
Table 3 gives an overview of <strong>the</strong> isolated pathogens, in which only <strong>the</strong> four marked fungi<br />
(Figure 31) have been used <strong>for</strong> all <strong>the</strong> experiments.<br />
Table 3: An overview of <strong>the</strong> isolated pathogens<br />
Code Source Isolated from<br />
Bra I<br />
Market<br />
Bra II Warakapola (Plantation)<br />
rambutan<br />
Bra III<br />
Market<br />
Bmu I Market Annona muricata<br />
Bsq I Medawachchiya<br />
Bsq II<br />
Market<br />
Annona squamosa<br />
Bsq III<br />
Market<br />
Bsq IV Market Annona squamosa ´Red`<br />
Results of <strong>the</strong> bio-assays:<br />
Figure 31: The four <strong>Botryodiplodia</strong><br />
<strong>sp</strong>. Isolates which were used <strong>for</strong> all<br />
experiments<br />
The bio-assay experiments have shown that three of <strong>the</strong> Trichoderma <strong>sp</strong>. isolates have a high,<br />
four a moderate and three a low activity against <strong>the</strong> pathogens, while three were observed as<br />
non effective (Table 4).<br />
33
Two of <strong>the</strong> Trichoderma <strong>sp</strong>. with a high effectiveness were isolated from rambutan<br />
plantations in Warakapola. The third of <strong>the</strong> high active ones was found in a soil sample taken<br />
from an Annona muricata tree in Wijayapura. The fungi which were isolated from soil<br />
samples taken from Annona squamosa trees showed <strong>the</strong> lowest activities against<br />
<strong>Botryodiplodia</strong> <strong>sp</strong>.. Only one of <strong>the</strong>se fungi had a moderate effectiveness.<br />
Table 4 presents <strong>the</strong> results of <strong>the</strong> bio-assay, whereas “+++” identifies a good, “++” a<br />
moderate, “+” a low and “0” no effectiveness against <strong>the</strong> pathogens.<br />
Table 4: Results of <strong>the</strong> effectiveness of <strong>the</strong> isolated Trichoderma <strong>sp</strong>.<br />
Code Source Activity<br />
Tra I Urapola ++<br />
Tra II Warakapola +++<br />
Tra III Warakapola +++<br />
Tsq I Matale 0<br />
Tsq II Matale +<br />
Tsq III Wijayapura ++<br />
Tsq IV Medawachchiya 0<br />
Tmu I Galagedera ++<br />
Tmu II Galagedera ++<br />
Tmu III Galagedera 0<br />
Tmu IV Matale +<br />
Results of <strong>the</strong> cross-inoculation experiment:<br />
To interpret <strong>the</strong> results of <strong>the</strong> experiments with <strong>the</strong>ir replications as overall results T-tests<br />
were undertaken. In all tests <strong>the</strong> null hypo<strong>the</strong>sis could be di<strong>sp</strong>roved.<br />
All pathogens have shown <strong>the</strong> same severity on <strong>the</strong> rambutan fruits after three days (Figure<br />
32). In <strong>the</strong> first two days <strong>the</strong> severity which was caused by <strong>the</strong> pathogen “Bmu I” was higher.<br />
Cross-inoculationNephelium lappaceum<br />
Severity [%]<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Day 1 Day 2 Day 3 Day 4 Day 5<br />
Time after inocculation [d]<br />
Bmu I Bsq IV Bra III Bsq III Control I Control II<br />
Figure 32: Results of <strong>the</strong> cross-inoculation on rambutan<br />
34
The evaluation of <strong>the</strong> cross-inoculation with Annona muricata showed that both controls have<br />
<strong>the</strong> same or a higher severity as <strong>the</strong> pathogens (Figure 33). On <strong>the</strong> fifth day of <strong>the</strong> experiment<br />
<strong>the</strong> severities of <strong>the</strong> pathogens Bmu I and Bsq III were higher than <strong>the</strong> ones of <strong>the</strong> o<strong>the</strong>rs and<br />
of <strong>the</strong> controls.<br />
Cross-inoculationAnnona muricata<br />
Severity [%]<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Day 1 Day 2 Day 3 Day 4 Day 5<br />
Time after inocculation [d]<br />
Bmu I Bsq IV Bra III Bsq III Control I Control II<br />
Figure 33: Results of <strong>the</strong> cross-inoculation experiment on Annona muricata<br />
As it is seen in Figure 34, <strong>the</strong> standard deviations of <strong>the</strong> results of <strong>the</strong> experiments with<br />
Annona squamosa fruits are high. Thus <strong>the</strong> results allow no exact interpretation.<br />
Cross-inoculation Annona squamosa<br />
Severity [%]<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Day 1 Day 2 Day 3 Day 4 Day 5<br />
Time after inocculation [d]<br />
Bmu I Bsq IV Bra III Bsq III Control I Control II<br />
Figure 34: Result of <strong>the</strong> cross-inoculation on Annona squamosa<br />
35
Results of <strong>the</strong> <strong>sp</strong>orulation experiment:<br />
Table 5 confirms results of <strong>the</strong> <strong>sp</strong>orulation experiment:<br />
Table 5: Results of <strong>the</strong> <strong>sp</strong>orulation experiment<br />
Pathogen<br />
Incubator<br />
Incubator and<br />
daylight<br />
Incubator, cold shock<br />
and daylight<br />
Bra II<br />
Sporulation after 24<br />
days<br />
Sporulation after 15<br />
days<br />
Sporulation after 26<br />
days<br />
Bmu I<br />
Sporulation after 24<br />
days<br />
Sporulation after 26<br />
days<br />
Bsq III<br />
Sporulation after 15<br />
days<br />
Sporulation after 26<br />
days<br />
Bsq IV<br />
Sporulation after 24<br />
days<br />
Sporulation after 26<br />
days<br />
The cold shock treatment of <strong>the</strong> cultures induced by all pathogens <strong>the</strong> longest <strong>sp</strong>orulation<br />
duration, at Bsq IV a <strong>sp</strong>orulation even failed to appear. The pathogens Bra II and Bsq II had<br />
<strong>the</strong> fastest <strong>sp</strong>orulation with <strong>the</strong> daylight treatment without a cold shock. Bsq IV and Bmu I<br />
<strong>sp</strong>orulated fastest in <strong>the</strong> incubator without any fur<strong>the</strong>r treatment. But it took <strong>the</strong>m with 24<br />
days <strong>the</strong> longest duration.<br />
36
10 Conclusion<br />
The results of <strong>the</strong> present study are showing that Trichoderma <strong>sp</strong>. are widely available in Sri<br />
Lanka and that some of those can be used as bio-control agents. SIVAKUMAR et al. (2000)<br />
isolated Trichoderma <strong>sp</strong>. from rambutan plantations and proved <strong>the</strong>ir antagonistic effects<br />
against <strong>Botryodiplodia</strong> <strong>the</strong>obromae as well.<br />
Only one of <strong>the</strong> bio-control agent (Bmu I) isolated from <strong>the</strong> surroundings of an Annona<br />
muricata tree was able to control <strong>the</strong> pathogen Bsq IV completely. This organism was<br />
isolated from a red skinned Annona squamosa. Consequently it seems that <strong>the</strong> Bio-control<br />
agent Bmu I is <strong>the</strong> most effective of all <strong>the</strong> BC-agents which were isolated <strong>for</strong> <strong>the</strong><br />
experiments.<br />
The results of <strong>the</strong> cross-inoculation experiment show that cross infections between rambutan<br />
und annona fruits can appear both in <strong>the</strong> field and during tran<strong>sp</strong>ortation and storage. Thus it is<br />
to recommend that <strong>the</strong>se fruits should not be tran<strong>sp</strong>orted or stored toge<strong>the</strong>r. It also underlines<br />
<strong>the</strong> importance of <strong>the</strong> site selection of new plantations <strong>for</strong> both fruits.<br />
The isolated Trichoderma <strong>sp</strong>. should be tested in fur<strong>the</strong>r studies in field experiments or in in<br />
vivo studies. For this, <strong>the</strong> BC-agent could be mixed into <strong>the</strong> soil or applied directly on <strong>the</strong><br />
plant or fruit.<br />
According to BRIMMER and BOLAND (2003) some Trichoderma <strong>sp</strong>. can have negative effects<br />
on o<strong>the</strong>r useful organisms. This should be investigated in fur<strong>the</strong>r studies as well.<br />
11 Acknowledgement<br />
I would like to thank <strong>the</strong> GTZ (Deutsche Gesellschaft für Technische Zusammenarbeit) <strong>for</strong><br />
supporting my stay in Sri Lanka by giving me a scholarship and Prof. Dr. Joachim Heller<br />
from <strong>the</strong> University of Applied Sciences of Geisenheim <strong>for</strong> his support.<br />
Fur<strong>the</strong>rmore I would like to express my sincere gratitude to Dr. Hannah Jaenicke, director of<br />
ICUC and Dr. Shanthi Wilson Wijeratnam, Manager of <strong>the</strong> Post Harvest Technology Group<br />
of ITI <strong>for</strong> giving me <strong>the</strong> possibility to attend my BPS in Sri Lanka.<br />
I am indebted to <strong>the</strong> whole Post Harvest Technology Group and to <strong>the</strong> ICUC team <strong>for</strong> giving<br />
me a very warm and hearty welcome in a complete new surrounding.<br />
I would like to give sincere thankfulness to Mrs. Nimali Abeyratne, senior officer of ITI and<br />
to Miss. Yashoda Dharmatilaka <strong>for</strong> <strong>the</strong>ir great encouragement during my work at ITI.<br />
37
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GEORGE, A.P. and NISSEN, R.J. (1991): Annona cherimola Miller, Annona squamosa L., A.<br />
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No. 2: Edible fruits and nuts. E.W.M. VERHEIJ and R.E. CORONEL (eds.), p. 71-75.<br />
GUPTA, V.P., TEWARI, S.K., GOVINDAIAH, P. and BAJPAI, A.K. (1999): Ultrastructure of<br />
Mycoparasitism of Trichoderma, Gliocladium and Laetisaria Species on <strong>Botryodiplodia</strong><br />
<strong>the</strong>obromae. Journal of Phytopathology, Vol. 147, p. 19-24.<br />
HARMAN, G.E. (2005): Trichoderma <strong>sp</strong>p., including T. harzianum, T. viride, T. konigie, T.<br />
harntaum and o<strong>the</strong>r <strong>sp</strong>p.. In: Biological Control: A Guide to Natural Enemies in North<br />
America. WEEDEN, R., SHELTON, H.S., LI, J. and HOFFMANN, T. (eds.), Cornell<br />
University, Geneva.<br />
http://www.nysaes.cornell.edu/ent/biocontrol/pathogens/trichoderma.html<br />
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Factsheet No. 5, Colombo: ICUC.<br />
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KORTING, F. (2001): Pilze ohne Chemie bekämpfen. Deutscher Gartenbau, Vol. 14, p. 33-34.<br />
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Different Per<strong>sp</strong>ective. J.E. HERNÁNDO BERMEJO and J. LEÓN (eds.), Plant Production<br />
and Protection Series No. 26. Rome: FAO, p. 85-92.<br />
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MORTON, J.F. (1987a): Rambutan. In: Fruits of warm climates, last update: 4/2/99, Miami:<br />
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39
ONG, P.K.C., ACREE, T.E. and LAVIN, E.H. (1998): Characterization of Volatiles in Rambutan<br />
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Fruit <strong>Crops</strong>. In: Diseases of Tropical Fruit <strong>Crops</strong>. (Eds) R.C. PLOETZ, CAP International,<br />
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40
13 Annexes<br />
Figure 35: Organisation structure of ITI (ITI, 2007)<br />
41
Figure 36: List of bio-control agents isolated<br />
Code Source Morphology Bio-control Activity Picture<br />
Bsq IV Bsq III Bra III Bmu I<br />
Tra I Urapola • Mycelial growth:<br />
(Madakotuwa) Covers <strong>the</strong> whole plate<br />
equal in 3 days after<br />
subculture.<br />
• Sporulation:<br />
Full <strong>sp</strong>orulation after 5<br />
days; plate is dark green<br />
coloured.<br />
Tra II Etena velle • Mycelial growth:<br />
Covers <strong>the</strong> plate with<br />
thick white mycelia in 3<br />
days after subculture and<br />
starts turning green in <strong>the</strong><br />
centre.<br />
• Sporulation:<br />
Full <strong>sp</strong>orulation after 5<br />
days with domination in<br />
<strong>the</strong> centre and <strong>the</strong> outer<br />
area.<br />
Tra III Etena velle • Mycelial growth:<br />
Covers <strong>the</strong> whole plate<br />
with a very thick white<br />
layer after 4 days, <strong>the</strong><br />
centre is yellowish-green<br />
coloured. After 6 days of<br />
incubation little black<br />
stone like balls are<br />
appearing.<br />
+ +++ +++ ++<br />
++ +++ +++ +++<br />
++ +++ +++ ++<br />
• Sporulation:<br />
Full <strong>sp</strong>orulation after 5<br />
days, but not homogeny<br />
coloured.<br />
Tsq I<br />
Matale<br />
(Home garden)<br />
• Mycelial growth:<br />
In 3 days after<br />
subculturing <strong>the</strong> whole<br />
plate is covered with a<br />
thin white mycelia, <strong>the</strong><br />
media in <strong>the</strong> centre is<br />
turning into an orangeyellowish<br />
colour.<br />
0 0 0 0<br />
• Sporulation:<br />
Full <strong>sp</strong>orulation after 6<br />
days but only less in <strong>the</strong><br />
centre and <strong>the</strong> outer area.<br />
42
Tsq II<br />
Matale<br />
(Homegarden)<br />
• Mycelial growth:<br />
Covers <strong>the</strong> full plate in 4<br />
days after subculturing.<br />
The colour is already light<br />
green.<br />
0 ++ ++ +<br />
• Sporulation:<br />
Full <strong>sp</strong>orulation after 5<br />
days, dark green colour.<br />
Tsq III Wijayapura • Mycelial growth:<br />
Mycelium is growing like<br />
many little dots which are<br />
<strong>sp</strong>read on <strong>the</strong> plate. After<br />
5 days <strong>the</strong> whole plate is<br />
covered with this little<br />
“mycelia dots”.<br />
• Sporulation:<br />
Complete <strong>sp</strong>orulation of<br />
<strong>the</strong> mycelia after 7 days.<br />
Tsq IV Medawachchiya • Mycelial growth:<br />
Mycelium is growing<br />
very heterogenic. After 4<br />
days <strong>the</strong> complete plate is<br />
covered. But <strong>the</strong> mycelia<br />
layer is not out of one<br />
piece. Spot like in <strong>the</strong><br />
outer part of <strong>the</strong> plate. It<br />
has a bad smell.<br />
+ +++ +++ ++<br />
0 0 0 0<br />
• Sporulation:<br />
Sporulation after 6 days.<br />
Tmu I Galagedera • Mycelial growth:<br />
Whole plate is covered in<br />
3 days after subculture<br />
with snow-white mycelia.<br />
After 8 days <strong>the</strong> mycelia<br />
starts to turn into a very<br />
light green.<br />
++ ++ + ++<br />
• Sporulation:<br />
Full <strong>sp</strong>orulation after 10<br />
days.<br />
Tmu II Galagedera • Mycelial growth:<br />
The plate is covered with<br />
white mycelia after 3<br />
days. The mycelia around<br />
<strong>the</strong> subcultered piece is<br />
turning dark green after 4<br />
days while <strong>the</strong> rest stays<br />
white till it turns light<br />
green after 5 days.<br />
++ ++ +++ ++<br />
• Sporulation:<br />
Sporulation of <strong>the</strong> full<br />
plate after 6 days after<br />
subculture<br />
43
Tmu III Galagedera • Mycelial growth:<br />
Covers <strong>the</strong> whole plate in<br />
4 days. Some parts of <strong>the</strong><br />
mycelia are turning <strong>the</strong><br />
colour into dark-green<br />
and some are staying<br />
white with a very thick<br />
mycelium-layer.<br />
• Sporulation:<br />
Full <strong>sp</strong>orulation after 6<br />
days, <strong>the</strong> white parts are<br />
getting a light greenyellowish<br />
colour.<br />
Tmu IV Matale • Mycelial growth:<br />
After 4 days <strong>the</strong> whole<br />
plate is covered with are<br />
white mycelia, except <strong>the</strong><br />
centre which green<br />
coloured. The surface of<br />
<strong>the</strong> mycelia is covered<br />
with many little craters.<br />
0 0 0 0<br />
++ ++ + 0<br />
• Sporulation:<br />
Full <strong>sp</strong>orulation after 6<br />
days, when <strong>the</strong> complete<br />
mycelia changed into a<br />
dark green colour.<br />
Tmu V Wijayapura • Mycelial growth:<br />
The mycelium never<br />
covers <strong>the</strong> whole plate<br />
and is not growing in a<br />
round way. After 4 days<br />
<strong>the</strong> mycelia changes into a<br />
light green colour.<br />
+++ ++ +++ +++<br />
• Sporulation:<br />
After 5 days with a very<br />
dark green colour.<br />
44