Effects of the Nematophagous Fungi Arthrobotrys oligospora Fresen and Trichoderma harzianum Rifai on Nematodes Infecting Banana, Tomato and Lime Plants
Suad Abdel Gamiel Mohamed Ahmed B.Sc. in Crop Protection Faculty of Agricultural Sciences University of Cairo, Egypt (1981)
M.Sc. in Crop Protection (Plant Pathology) Faculty of Agricultural Sciences University of Gezira, (1997)
A Thesis Submitted to the University of Gezira in Fulfilment of the Requirements for the Award of the Degree of Doctor of Philosophy in Crop Protection (Plant Pathology) Plant Protection Department Faculty of Agricultural Sciences University of Gezira
September, 2013
Effects of the Nematophagous Fungi Arthrobotrys oligospora (Fresen) and
Trichoderma harzianum (Rifai) on Nematodes Infecting Banana, Tomato and Lime Plants
By
Suad Abdel Gamiel Mohamed Ahmed
Supervision committee:
Name Position Signature
Prof. El Nour El Amin Abdel Rahman Main Supervisor …………
Prof. Ahmed El Bashir Mohamed El Hassan Co-Supervisor …………
Date: September, 2013
Effects of the Nematophagous Fungi Arthrobotrys oligospora (Fresen) and Trichoderma harzianum (Rifai) on Nematodes Infecting Banana, Tomato and Lime Plants
By
Suad Abdel Gamiel Mohamed Ahmed
Examination Committee:
Name Position signature
Prof. El Nour El Amin Abdel Rahman Chairman ………………
Prof. Abel Mageed Yassin Abel Mageed External Examinar………………
Dr. Mohamed Hamza Zain Alabdeen Internal Examiner………………
Date of Examination: 29, September, 2013
DEDICATION
To my beloved and kind family, my late dear father, my late dear mother,
Brothers, sisters, my late dear sister Sharifa and late dear brothers Mohamed and Ahmed. To my kind husband El Badri Yassin
ACKNOWLEDGEMENT
First of all my thanks to the Almighty ALLAh who gave me strength ,power and patience to complete this study and bring it to its end.
My sincere gratitude , appreciation and a lot of thanks to my supervisor Prof. El Nour El Amin for his keen guidance, interest . kind close supervision and encouragement. I am also grateful to my co-supervisor Dr. Ahmed Elbashier Mohamed Elhassan for hidiscussions during the progress of the study. Special gratitude and appreciation are due to the staff of plant pathology section and plant pathology lab. for they had always assisted me during my lab. work. Special thanks also due to pesticides residue lab. , specially Mr. Saeed Hamid for the very valuable help and assistance. Deep sincere gratitude to staff of Nematology lab. specially Mr. Sir El Khatim Momhamed, Iman and Samia Yousif for their valuable assistance during preparing, washing and counting nematodes under study. I am deeply indebted to Pof. Dafalla Ahmed Dawoud for his good ideas and advises. I am extremely grateful to my family for their continual encouragement and strong support.
My sincere thanks and deep appreciation are extended to my faithful friends and colleagues for their valuable encouragement and assistance offered during the course of this study.
My greatest appreciations to any unnamed person , who contributed in one way or another at time of difficulties.
I am indebted to the Agricultural Research Corporation for the unlimited support and for giving me the chance to do this work. My appreciation and gratitude are extended to the government of Sudan for funding and financially supporting this study.
Last but not least I wish to express my thanks and appreciative acknowledgement to my husband El Badri for his sacrifice and moral support.
Effects of the Nematophagous Fungi Arthrobotrys oligospora Fresen and Trichoderma harzianum Rifai on Nematodes infecting Banana, Tomato and Lime Plants
Doctor of Philosophy in Plant Protection(Plant Pathology) (September, 2013)
Department of Crop Protection
Faculty of Agriculture Sciences, University of Gezira
ABSTRACT
Nematophagous fungi are the fungi which attack nematodes, many of them are plant pathogens. These fungi worldwide in distribution and have been reported from many countries This study has been carried out to search for nematophagous fungi in Gezira including Sudan. soil and their capability to attack nematodes. Random samples collected from Gezira soil grown with tomato, banana and lemon crops were put on Corn Meal Ajar media (CMA) for the growth of the fungi and nematodes. Using Digital Microscope many types of trapping nematodes had been seen, Such as those with adhesive nets, adhesive knob and presence of fungal spores The samples labeled, and kept in laboratory for further inside the dead body of the nematode. The fungi has been identified as Arthrobotrys oligospora and .study in the glass house Trichoderma harzianum. The study investigated the capability of those fungi to attack and destroy nematodes. The nematophagous fungi A. oligospora was found to attack the nematode similis It was also noticed that, the nematode had been captured by adhesive knops Radophilus and after that the nematode struggled until death. The fungus A. oligospora penetrates the nematode cuticle and consumed all the body content of the nematode. Also the same fungus had been seen capturing the nematode Xiphenema sp. by adhesive net and had been held at two points and sometimes at several points. The fungus T. harzianum has been tested for its potential as a biological control agent and was found to suppress nematode population densities when it was added in different concentrations (105, 104 and103 spores/ml) to 50 Meloidojyne javanica nematodes in soil planted with tomato. The fungus reduced the numbers of nematode galls on tomato roots compared with the untreated control and resulted in excellent plant vegetative growth. Growing of lemon plants on soil artificially infested with Xiphenema sp. nematodes and treated with different concentrations of the fungus A. oligospora inocula significantly increased the number of lemon plant leaves, stem length, root length and also increased the fresh and dry root weight compared to the same soil without fungal inoculation. Also the different concentrations of the same fungus resulted in excellent vegetative growth of banana plants when grown in soil artificially infested with the nematode R. similis. Effect of different rates of the fungus T. harzianum metabolites on the nematode Xiphenema sp. had been studied. The different rates of the fungal metabolites (10-1, 10-2 and 10-3) after 24 hours gave comparable percentage of mortality, 16.8%, 14.19% and 11.65% respectively, but significantly better than the untreated control that resulted in 3.78% mortality. After 72 hours, the highest concentrations of the fungal metabolites resulted in significantly high nematodes mortality (66.24%) compared with the lowest one (42 93%) and the untreated control (8.87%). The study recommended that, Nematophagous fungi, if given more attention may be useful as biological control which can decrease cost of nematicides and conserve the ecosystem.
على النيماتودا التى تصيب .harzianum Rifai T وA. oligospora Fresenتأثير الفطريات القاتله للنيماتودا
نباتات الموز، الطماطم و الليمون
دكتوراة الفلسفة في وقاية المحاصيل )امراض النبات( قسم وقاية المحاصيل، كلية العلوم الزراعية، جامعة الجزيرة
ملخص الدراسة الفطريات القاتله للنيماتودا هي تلك التي تهاجم النيماتودا والتي كثيرا منها مسببا المراض النبات. وهذه الفطريات كثيرة االنتشار وسجلت قي كثير من البلدان بما فيها السودان. أجريت هذه الدراسه للبحث عن بعض الفطريات التي تقوم بمهاجمة انواع مختلفة من النيماتودا. جمعت عينات عشوائية من مناطق مختلفة بالجزيرة زرعت بمحاصيل الطماطم,الموز والليمون. وضعت العينات في بيئة مناسبة لنمو الفطر والنيماتودا معا. باستخدام المجهر الرقمي ,شوهدت عديد من طرق هجوم الفطر علي النيماتودا, منها صنع الفطر لشبكه من الميسيليوم واصطياد النيماتودا بها, أيضا هناك بعض الفطريات تقوم بعمل حلقات منقبضة و غير منقبضة وتعمل علي خنق النيماتودا بها حتي تموت. فطريات أخري تخترق جدار النيماتودا وتقوم باستهالك األحشاء الداخلية للنيماتودا وتكوين جراثيمها داخل جسم النيماتودا. تم التعرف علي هذه الفطريات بعد عزلها بالمعمل ,كما تم التعرف علي النيماتودا أيضا وتم حفظ العينات بالمعمل وذلك إلجراء الدراسة الالزمة بالمشتل. الفطريات التي تم التعرف عليها هي , Arthrobotrys oligosporaو Trichoderma harzianum. خالل هذه الدراسة تمت معرفة مدي مقدرة تلك الفطريات علي مكافحة الديدان ألثعبانيه. لغرض هذه الدراسة تم جمع عينات التربة من مناطق مختلفة هي مزرعة هيئة البحوث الزراعيه, منطقة باشكار )قسم المسلميه(, وادي شعير, أم سنط و حنتوب. اوضحت هذه الدراسه النشاط العالي للفطرA. oligospora علي مهاجمة بعض الديدان الثعبانيه خاصة Radophillus similis المعروفه بمقدرتها علي مهاجمة نبات الموز وتؤدي الي نقصان اال نتاجيه. اوضحت الدراسه ان الديدان الثعبانيه تقبض بالتصاقها بالحلقات المنقبضه وتستمر الديدان الثعبانيه بالمقاومه حتي تموت. الفطر A. oligospora يخترق جدار الديدان الثعبانيه ويستهلك كل محتوياتها. كذلك يصطاد هذا الفطر الديدان الثعبانيه من نوع .Xiphenema sp ويلتصق بها بواسطة شبكه في اكثر من نقطه. وهذا النوع من الديدان الثعبانيه معروف بمهاجمته الشجار الليمون. الفطر .T harzianum تم اختياره في مجال المكافحه الحيويه كعامل مؤثر علي تقليل كثافة الديدان الثعبانيه Meloidojyne javanica عندما اضيف بتركيزات مختلفه and 103) spores/ml 104 ,105( مع عدد 50 من هذه الديدان في تربه زرعت بنباتات طماطم. اوضحت النتائج مقدرة هذا الفطر علي تقليل االنتفاخات في جذور الطماطم مقارنة بالشاهد كما اوضحت النتائج مقدرة هذا الفطر في تحسين النمو الخضري للنبات مما يوضح مقدرته فى مكافحة الديدان الثعبانيه . أدت اضافة الفطر A. oligospora بتركيزات مختلقه ) (and 10-3 2-10 ,1-10) مع عدد 50 من الديدان الثعبانيه Xiphenema sp الي التربه الي زيادة أوراق نبات الليمون ,طول السيقان, طول الجذور و الوزن الجاف والرطب للجذور لموسمين مقارنة مع الشاهد. كذلك أدي نفس الفطر عندما أضيف بتركيزات مختلفة and 10- 10-2 ,10-1) (3 مع عدد 50 من الديدان الثعبانيه R. similis. الي تربه زرعت بنبات الموز لتحسين النمو الخضرى للنبات من زياده لعدد االوراق,طول السيقان وزيادة الوزن الجاف والرطب للجذور.أدي وضع تركيزات مختلفه من metabolites الفطر T. harzianum (3-10 2-10 ,1-10) مع أعداد معينه من الديدان الثعبانيه .Xiphenema sp في أطباق بتري الى نسبه عاليه من قتل الديدان الثعبانيه تراوحت بين 11.65%- 16.8% بعد 24 ساعه مقارنة مع 3.78% للشاهد وتم الحصول علي نفس النتائج بعد 48 و 72 ساعه. ادي اعلي تركيز إلي نسبة موت بلغت 66.24% بينما أدي التركيز األقل والشاهد الي 42.93% و 8.87% علي التوالي. توصي الدراسه بانه اذا ما وجدت الفطريات القاتله للنيماتودا مزيدا من االهتمام يمكن ان تفيد في المكافحه الحيويه وتقلل من تكلفة المبيدات النيماتوديه وتحمي المنظومه البيئيه.
LIST OF CONTENTS
page
DEDICATION……………………………………………………………………… ………………………………..iii ACKNOWLEDGEMENT……………………………………………………… ………………………………iv ABSTRACT………………………………………………………………………… ………………………………….v ARABIC ABSTRACT………………………………………………………………………… …………………vi LIST OF CONTENTS………………………………………………………………………… ………………...vii LIST OF TABLES……………………………………………………………………………… …………………..xiv LIST OF FIGURES………………………………………………………………………………………………..xvii
CHAPTER ONE : INTRODUCTION…………………………………………………………………….1 Introduction…..…………………………………………………………………………………………….……….1
CHAPTER TWO : LITERATURE REVIEW…………………………………………………………..5
2.1. Definition of nematophagous fungi…………………………………………………………….……..5
2.2. Ecological aspects of nematode suppression by fungi……………………………….………8
2.2.1. Distribution and habitat of nematophagous fungi………………………………….……….8
2.2.2. Nutritional adaptation of nematophagous fungi…………………………………….………10
2.2.3. Interaction of nematophagous fungi with host nematode……………………………..13
2.2.4. Interaction of fungal antagonists of nematodes with other soil organism……...15
2.2.5. Effects of plant and cropping system…………………………………………………………..17
2.3. Effects of soil a biotic factors……………………………………………………………………………..19
2.3.1. Soil temperature………………………………………………………………………………………….…19
2.3.2. Soil moisture……………………………………………………………………………………………….….20
2.3.3. Soil texture………………………………………………………………………………………………………21
2.3.4. Effect of soil pH, nutrients, organic matter, and Agricultural chemicals……..……22
2.4. Biological control of nematodes by fungal antagonists…………………………………….…24
2.5. Type of fungal antagonists of nematode…………………………………………………………….25
2.5.1. Predacious fungi………………………………………………………………………………………..…..25
2.5.1.1. Fungi with adhesive hyphae…………………………………………………………………………26
2.5.1.2. Fungi with adhesive branches…………………………………………………………………….…26
2.5.1.3. Fungi with adhesive nets…………………………………………………………………………….…27
2.5.1.4. Fungi adhesive knops……………………………………………………………………………………27
2.5.1.5. Fungi with non constricting rings………………………………………………………………….28
2.5.1.6. Fungi with constricting rings…………………………………………………………………….…..28
2.5.1.7. Stephano cysts………………………………………………………………………………………….…..29 2.5.2. Endoparasites of vermiform nematodes…………………………………………………..……..30
2.5.2.1. Obligate endoparasites of vermiform nematodes………………………………………...30
2.5.2.1.1. Encysting species……………………………………………………………………………………...30
2.5.2.1.2. Species forming adhesive conidia……………………………………………………………...31
2.5.2.1.3. Species with conidia that may be ingested…………………………………………….....31
2.5.2.1.4. Species with gun cell………………………………………………………………………………...32
2.5.2.2. Facultative parasites of vermiform nematodes……………………………………….……32
2.5.2.3. Parasites of sedentary females and eggs……………………………………………….…….33
2.5.2.3.1. Obligate parasites of sedentary females and eggs…………………………….……….33
2.5.2.3.2. Facultative parasites of eggs and sedentary females…………………………………34
2.5.2.4. Fungi producing antibiotic substance………………………………………………………....35
2.5.2.5. Vesicular – arbuscular mycorrhizel …………………………………………………………….36
2.6. Modes of infection of nematodes of nematodes……………………………………………….37
2.6.1. Attraction………………………………………………………………………………………………………38
2.6.2. Attachment……………………………………………………………………………………………………38
2.6.3. Penetration……………………………………………………………………………………………………39
2.6.4. Pathogenicity of fungi to nematode……………………………………………………………….42
2.7. Suppressive soil associated with fungal antagonists…………………………………………44
2.7.1. Decline of Heteodera avenae in Europe………………………………………………………..45
2.7.2. Suppression of Heteodera avenae in Europe…………………………………………………45
2.7.3. Suppression of Heteodera schachtii by fungi…………………………………………………47
2.7.4. Suppression of Meloidogyne spp. by Dactylella oviparasitica…………………………48
2.7.5. Suppression of Mesocrisonema xenoplax by Hirsutella rhossiliensis………………48
2.8. Potential fungal agents for control of nematodes…………………………………………….49 2.8.1. Paecilomyces lilacinus…………………………………………………………………………………..50
2.8.2. Verticillium chlamydosporum………………………………………………………………………..52
2.8.3. Verticillium lecanii………………………………………………………………………………………….52
2.8.4. Haustella rhossiliensis…………………………………………………………………………………...55
2.8.5. Fusarium spp………………………………………………………………………………………………...57
2.9. Ecology of nematophagous fungi……………………………………………………………………..58
2.10. Taxonomy of nematophagous fungi………………………………………………………………,59
2.11. Biology of nematode – destroying fungi………………………………………………………..60
2.11.1. Spores………………………………………………………………………………………………………...60
2.12. Biological control…………………………………………………………………………………………..62
2.13. Chemical attractants……………………………………………………………………………………..64
2.14. Specificity………………………………………………………………………………………………………67
CHAPTER THREE : MATERIALS AND METHODS …………………………………….70
3.1. Sites localities………………………………………………………………………………………………….70
3.1.1. Sampling method………………………………………………………………………………………….70
3.2. Method of isolation of soil fungi ……………………………………………………………………..70
3.2.1. Media used…………………………………………………………………………………………………..71
3.2.2. Examination of plates and recovery of fungal isolates and nematodes………….71
3.2.3. Isolation of fungi from around the captured nematode…………………………………71
3.3. Identification of the isolates…………………………………………………………………………….72
3.3.1. Identification of Trichoderma harzianum isolates………………………………………….72
3.3.2. Identification of Arthrobotrys oligospora isolates……………………………………...... 72
3.4. preparation of the fungal inocula…………………………………………………………………….72
3.4.1. preparation of the fungus Trichoderma harzianum and counting the spores..72 3.4.2. Preparation of the fungus Arthrobotrys oligospora and counting the spores…73
3.5. Preparation of nematode………………………………………………………………………………..73
3. 5. 1. Collection of infested soil samples……………………………………………………………….73
3.5.2. Washing and extraction of nematode…………………………………………………………..74
3.5.3. Estimation of living nematodes…………………………………………………………………....74
3.6. Glasshouse experiments………………………………………………………………………………….74
3.6.1. Experiment (1)…………………………………………………………………………………………….74
3.6.2. Experiment (2)…………………………………………………………………………………………….75
3.6.3. Experiment (3)……………………………………………………………………………………………..75
3.7. Metabolites of the fungus Trichoderma harzianum………………………………………...76
3.7.1. Medium preparation and inoculums…………………………………………………………….76
3.7.2. Purification of the metabolites…………………………………………………………………….77
3.7.3. Effects of the metabolites on the nematode………………………………………………….77
CHAPTER FOUR : RESULTS……………………………………………………………………………....78
4.1.Preliminary examination and observations of nematophagous fungi before identification……………………………………………………………………………………………………………78
4.1.1. Isolation of fungi from around the nematodes……………………………………………..84
4.1.2. Identification of Trichoderma isolates…………………………………………………………..84
4.1.3. Identification of Arthrobotrys isolates…………………………………………………………..84
4.2 Preparation of nematode under test………………………………………………………………..85
4.3. Glass house experiment……………………………………………………………………………………85
4.3.1. Experiment (1)……………………………………………………………………………………………...85
4.3.1.1. Effect of different concentrations of the fungus Trichoderma inocula on number of tomato leaves…………………………………………………………………………………..85
4.3.1.2. Effect of different concentrations of the fungus Trichoderma inocula
on stem length of tomato……………………………………………………………………………………….87
4.3.1.3. Effect of different concentrations of the fungus Trichoderma inocula
on tomato root length, fresh root weight and dry root weight……………………………….88
4.3.1.4. Effect of the fungus inoculums on number of nematode in the soil which is related to tomato after removal of plant under test…………………………...90
4.3.2. Experiment (2)………………………………………………………………………………………………92
4.3.2.1. Effect of different concentrations of the fungus Trichoderma
inocula on number of Lemon leaves………………………………………………………………………92
4.3.2.2. Effect of different concentrations of the fungus A. oligospora inocula on lemon stem length………………………………………………………………………………94
4.3.2.3. Effect of different concentrations of the fungus A. oligospora
inocula on lemon root length, fresh root weight and dry root weight………………….96
4.3.2.4. Effect of the fungus inocula on number of nematode in the soil which is related to lemon after removal of plant under test……………………….98
4.3.3. Experiment (3)………………………………………………………………………………………….99
4.3.3.1. Effect of different concentrations of the fungus A. oligospora
inocula on number of banana leaves…………………………………………………………………99
4.3.3.2. Effect of different concentrations of the fungus A. oligospora inocula on banana stem length…………………………………………………………………………100
4.3.3.3. Effect of different concentrations of the fungus A. oligospora
inocula on banana root length, fresh root weight and dry root weight……………..101
4.3.3.4. Effect of the fungus inocula on number of nematodes
in the soil which is related to banana after removal of plant under test..... …..103
4.4. Effects of T. harzianum metabolites on the nematode………………………………105
5. CHAPTER FIVE : DISCUSSION……………………………………………………………….106
CONCLUSIONS……………………………………………………………………………………………..113
Recommendations……………………………………………………………………………………..115
REFERENCES…………………………………………………………………………………………………116
LIST OF TABLES
Table No. page
1 Effect of different concentrations of the fungus T. harzianum inocula on
number of tomato leaves season (1) ………………………………………………………………….85
2 Effect of different concentrations of the fungus T. harzianum inocula on
number of tomato leaves season (2)………………………………………………………………… 86
3 Effect of different concentrations of the fungus T. harzianum inocula on
stem length of tomato season (1)…………………………………………………...... 87
4 Effect of different concentrations of the fungus T. harzianum inocula on stem length of tomato season (2)…………………………………………………………………….. 88
5 Effect of different concentrations of the fungus T. harzianum inocula on
stem length of tomato season (2)………………………………………………………………89
6 Effect of different concentrations of the fungus T. harzianum inocula on
tomato root length, fresh and dry root weight, season (2)………………………………….89
7 Effect of the fungus T. harzianum inocula on number of nematode in the
soil after removal of plant under test, season (1)………………………………………………..91
8 Effect of the fungus T. harzianum inocula on number of nematode in the
soil after removal of plant under test, season (2)……………………………………………….91
9 Effect of different concentrations of the fungus A. oligospora inocula on
number of Lemon leaves, season (1)…………………………………………………………………….93
10 Effect of different concentrations of the fungus A. oligospora inocula on
lemon leaves, season (2)…………………………………………………………………………………….93
11 Effect of different concentrations of the fungus A. oligospora inocula on
lemon stem length, season (1)……………………………………………………………………………95
12 Effect of different concentrations of the fungus A. oligospora inocula on
lemon stem length, season (2)……………………………………………………………………………95
13 Effect of different concentrations of the fungus A. oligospora inocula on
lemon root length, fresh root weight and dry root weight, season (1)………………..97
14 Effect of different concentrations of the fungus A. oligospora inocula on lemon root length, fresh root weight and dry root weight, season (2)……………….97
15 Effect of the fungus A. oligospora inocula on number of nematode in the
soil after removal of plant under test, season(1) ………………………………………………..98
16 Effect of the fungus A. oligospora inocula on number of nematode in the
soil after removal of plant under test, season (2)………………………………………………..98
17 Effect of different concentrations of the fungus A. oligospora inocula on
number of banana leaves, season (1)…………………………………………………………………99
18 Effect of different concentrations of the fungus A. oligospora inocula on
number of banana leaves, season (2)………………………………………………………………..99
19 Effect of different concentrations of the fungus A. oligospora inocula on
banana stem length, season (1)…………………………………………………………………………..100
20 Effect of different concentrations of the fungus A. oligospora inocula on
banana stem length, season (2)………………………………………………………………………100
21 Effect of different concentrations of the fungus A. oligospora inocula on
banana fresh root weight and dry root weight, season (1)………………………………102
22 Effect of different concentrations of the fungus A. oligospora inocula on
banana fresh root weight and dry root weight season (2)……………………………..102
23 Effect of the fungus A. oligospora inocula on number of nematode in the
soil after removal of plant under test, season (1)……………………………………………104 24 Effect of the fungus A. oligospora inocula on number of nematode in the soil
after removal of plant under test, season (2)………………………………………………….104
25 Percentage of mortality………………………………………………………………………………….105
LIST OF FIGURE
Figure page
1 Nematode captured by adhesive net and had been held at two points…………..79
2 Nematode trapped by net and had been attached at several points……………….79
3 Nematode captured by adhesive knob, the knob is seen left of picture………….80
4 The nematode struggled after capturing and dead…………………………………………80
5 The spores of A. oligospora inside the dead body of the nematode……………....81
6 The mycelium and spores of the fungus A. oligospora……………………………………81 7 The mycelium inside the host body and all the body contents had been
consumed ………………………………………………………………………………………………………..82
8 Nematode on agar plate dead with the toxin of Trichoderma………………………...82
9 Nematode on agar plate dead with the toxin of Trichoderma………………………...83
10 Spores penetrating through host cuticle……………………………………………………….83
11 Inside the host body had been consumed by the fungus and the spores
arise out of the host corpus………………………………………………………………………….84
12 The effect of different concentrations of the fungus T. harzianum inocula on the vegetative growth of the tomato plants……...... 86
13 Effect of T. harzianum inocula on the incidence of root knot nematode
on tomato……………………………………………………………………………………………………90
14 Effect of different concentrations of T. harzianum inocula on number of galls on tomato plant roots……………………………………………………………………...... 92
15 Effect of the different concentration of A. oligospora inocula on the vegetative growth of Lemon plants……………………………………………………………………..94
16 Effect of the high concentration of A. oligospora inocula on the number
of lemon leaves and stem length……………………………………………………………………… 96
17 Effect of the different concentrations of A. oligospora inocula on the vegetative growth of banana plants…………………………………………………………………..101
18 Effect of the high concentration of A. oligospora inocula on the number
of banana leaves and stem length………………………………………………...... 103 19 Effect of the high concentration of A. oligospora inocula on banana root weight………………………………………………………………………………………………………………104
CHAPTER ONE
INTRODUCTION
The nematodes attacked by fungi are cosmopolitan in occurrence as dominant members of the micro fauna in soil and organic debris. The numbers of nematodes in microbiologically active soils are estimated to range from one to twenty million individuals per square meter. Nematodes are often referred to as eel- worms. In size, nematodes are quite tiny, usually measuring about 100 – 1000 um in length although in some species they may be several mm. In nature nematodes may be parasitic on plants or animals and many are predatory on other species of nematodes. The majority, however, are the so- called free-living nematodes that feed largely by ingesting bacteria or fungal spores. Many nematophagous fungi exhibit remarkable specialization for their parasitic or predatory habits. Based on their mode of infection, they may be classified as either endo parasite or predatory. Endo-parasites produce conidia which are ingested by the nematode or attach to its external surface. Others infect eggs by invasive growth of mycelium. Predatory fungi produce specialized hyphae which trap nematodes and secure them during infection. All organisms in an ecosystem are influenced by abiotic and biotic factors. Nematodes are not exception. In an undistributed ecosystem, many nematode populations might be at equilibrium. When humans introduced agriculture into an ecosystem, the equilibrium might be broken and community structure might be changed dramatically so that some nematodes became severe pests of cultivated crops. Nevertheless, these agricultural pests are continuously subjected to attack by a number of natural enemies. The organisms that have adverse effects on nematode populations are collectively called nematode antagonists. The action of antagonists in maintaining nematode population density at a lower average than would occur in their absence can be called biological control. Biological control is the action of living organisms as pest control agents. It may variously defined as an applied field of endeavor or as a natural phenomenon. In the applied sense, it may be defined simply as the utilization of natural enemies to reduce the damage caused by noxious organisms to tolerable level. Soil-borne pests and diseases are inherently difficult to control. Biological control of nematodes was proposed as soon as plant-parasitic nematodes were recognized as devastating plant pathogens. Also it was largely neglected in the 20th-century. The discovery of chemical nematicides provided a cost effective and often spectacular means for nematode control consequently, chemical nematicides dominated nematode control in the past century. Even though they were environmentally and ecologically dangerous . Chemical treatments are applied to some high value crops. Reports of health and environmental hazards associated with their intensive use has been stated. Some commercial companies have identified soil-born pests and the diseases promising target for biological control. Nematophagous fungi are worldwide in distribution and have been reported from many countries . Virtually nematophagous fungi occurred wherever nematodes can live. The distribution, however differ among species of nematophagous fungi. Many predacious fungi and facultative parasitic fungi are widely distributed in the world. Some species however are distributed in a limited geographic area. Knowledge in this area begins at more than one hundred and twenty five years ago with Fresenius (1852) who was studying fungi on organic debris. His attention was attracted by a fungus producing scattered conidiophores over the surface of the substrate, each conidiophore produced clusters of large, two celled conidia giving a nodded appearance. Freseninus named this fungus Arthrobotrys oligospora. Some years later Woronin (1870) found that when the conidia of the fungus were germinated on old manure, certain of the aerial hyphae recurred and fused with themselves to form net- like tails whose function was not known. Zopf (1888) found that actively motile where the nematodes are captured by intargelement and here they struggle to free themselves and they stop feeding till they ultimately die. Barron (1977) in his book classified the nematode destroying fungi as either predatory or endoparasitic, and defined the endoparasitic fungi as those fungi which form spores which stick to the passing nematodes. Once these spores hatch, the sporoplasm enters the body and consumes its content to form a mass of hyphae Barron also included the egg parasites as special group of endoparasites specialized in the parasitism of nematode eggs. He classified them in the following way : those which possess :- 1- Adhesive hyphae 2-Ahesive branches 3- Adhesive nets 4-Adhesive knobs 5- Constricting rings 6- Non- constricting rings However, Mankau (1980) in his review stated that the antagonists of nematodes consisted of : 1- Nematode trapping fungi 2- Endoparasitic fungi 3- Parasites of eggs 4- Parasites of cyst nematodes 5- Fungi that produce metabolites toxic to nematodes All predators are present in the soil and they can propagate as saprophytes and their propagules are found as spores or fragments of hyphae.
Endozoic nematophagous fungi, on the other hand, are present in the soil only as spores which are swolled by nematodes or adhere the nematode corpus and they grow penetrating the corpus and consume the whole body of the nematode and produce their spores outside. Most of predators and certain of the endoparasites are not specific, and any particular fungus may attack a wide range of nematode species. It is natural therefore that nematode destroying fungi should be considered as agents for biological control. The main objective of the study is to:- 1- Survey soil types for detecting the presence of nematophagous fungi. 2- To select the most damaging nematodes for laboratory tests against test organisms that are known to be pathogenic to crop plants and vegetables like tomato, banana , carrot and lemon (free living and parasitic nematodes).
CHAPTER TWO
LITERATURE REVIEW
2.1. Definition of nematophagous fungi :-
Nematophagous fungi are fungi that feed on nematodes. These fungi can be egg parasitic. Nematode-trapping fungi are capable of making hyphae or proliferate in the soil in absence of nematodes and form traps in petri dish culture. They capture their prey by adhesive hyphae, adhesive knops, two dimensional traps, three dimensional traps and constricting or non constricting rings. They penetrate the corpus of the nematode and consume the whole body. Endo parasitic fungi, on the other hand, present in the soil as spores only, if they are swallowed or adhere to the corpus they will develop there, filling the hall body and sporulate outside. There are various ways for soil-borne fungi to suppress nematode multiplication. A detailed review of fungi as biocontrol agents against plant-parasitic nematodes has been published (Kerry and Jaffee, 1997). In summary, there are five mechanisms that fungi used to suppress nematodes. Some of these interactions are direct whereas others are indirect. The direct mechanism is performed by, 1) fungi that feed on nematodes directly, known as nematophagous fungi; 2) fungi interact with a nematode that kill nematodes by mycotoxins ( Baron and Thorne,1987) or 3) through the destruction of the feeding sites of sedentary nematodes in roots, (Glawe and Stiles,1989); 4) fungi that are nonpathogenic to plants, but compete with nematodes in roots and significantly reduce nematode multiplication (Sikora, 1992). Many of these fungi are used as potential nematode biocontrol agents; 5) Mycorrhizal fungi improve the growth of nematode infected plant and may also affect nematode development (Hussey and Roncadori, 1982). According to a survey of nematophagous fungi Ireland by Gray (1983), nematophagous fungi were found in all of the habitats examined, among these, permanent pasture, coniferous leaf litter, and the coastal vegetation while had the most frequent incidence of nematophagous fungi. Other habitats examined by Gray included old and partly revegetated dung, permanent grassland pasture, cultivated land, moss cushions, decaying vegetation and compost, and peatland (Gray, 1983). In addition, many other studies also supported the hypothesis that nematophagous fungi are widely distributed and have grate potential to be explored as biocontrol agents (Barron,1977). According to their feeding habits nematophagous fungi summarizes to many groups:- A) Nematode- trapping fungi Facultative fungi that form trapping structure to trap nematodes. There are six types of traps reported by Barron (1977). A) Nematode-trapping fungi; Facultative fungi that form trapping structure to trap nematodes. There are six type of traps reported by Barron (1977). 1- Adhesive hyphae: present in some genera of Class Zygomycetes eg. Stylopage and Cystopage 2- Adhesive traps: present in some genera of Class Deuteromycetes eg. Monacrosporium cionopagum (branches) M. ellipsosporum (knops) Arthobotrys oligospora (networks) 3- Non-adhesive traps: present in some genera of Class Deuteromycetes eg. Arthobotrys dactyloides (constricting ring) Dactylella leptospora (non- constricting ring) B) Facultative parasitic fungi attacking sedentary stages of nematode (Kerry and Jaffi,1997) These are facultative fungi that are commonly soil saprophytes, and are opportunistic fungi isolated from the sedentary stages (female and egg stages) of sedentary nematodes such as Heterodera, Globodera and Meloidogyne. They do not form specialized infection structures except appressoria. They can survive and proliferate in soil in the absence of nematodes, this present in the genera of class Hyphomycotina eg. Acremonium, Fusarium, Paecilomyces and Verticillium. C) Endoparasitic fungi (keryy and Jaffi, 1997). These are obligate parasitic fungi that have limited growth in soil outside the colonized nematode cadaver. * They can infect vermiform nematodes by producing adhesive spores attached to cuticle of passing nematodes, this can be found in genera of Hyphomycotina eg. Verticillium sp.and Hirsutella rhossiliensis. * Some can infect vermiform nematodes by producing conidia spores that can be ingested by nematodes eg. Harposporium anguillulae Some can infect vermiform nematodes by producing motile zoospores that encyst on the nematode surface, this present in Class Oomycetes eg. Myzocytium spp. and Lagenidium spp. *Some can infect sedentary nematodes when the nematodes were exposed on the root surface. this present in Class Oomycetes eg. Nematophthora gynophila. 2. 2. Ecological aspects of nematode suppression by fungi:-
2.2.1. Distribution and habitat of nematophagous fungi:-
Nematophagous fungi are world wide in distribution and have been reported from many countries (Gray, 1987). Virtually, nematophagous fungi occur wherever nematodes can live. The distributions, however, differ among species of nematophagous fungi. Many predacious fungi and facultative parasitic fungi are widely distributed in the world. Some species, however, are distributed in a limited geographic area. In Britain and Ireland, the most frequently recorded endoparasites of nematodes were Acrostalagmus obovatus and H. anguillulae, and the most common predacious fungi were Dactylella bembicoids, D. ellipsospora and D. cionopaga (Duddington, 1951; Gray,1983). The species of endoparasitic fungus, Harposporium helicoids, was frequently isolated in Ontario, Canada (Barron,1978), but rarely in Ireland (Kerry and Andersson,1983). While the distributions of Dactylella mammillata in Britain, A. oligospora in Ireland and A. musiformis. In both Ireland and Britain appear highly restricted, all species are locally abundant (Gray,1987). Leu et al. , (1993) reported that the predacious fungi that were the most widely distributed in China include Monacrosporium thaumasium, M. eudermatum, A. oligospora, A. conoides, A. robusta, A. oviformis, and A. brochopaga. M. thaumasium was frequently encountered in the temperate areas in China. A. oligospora was most abundant in temperate soils in some surveys but not in other studies (Gray, 1983; Liu, et al. ,1993). Studies of the species and frequency of fungal parasites of females and eggs of sedentary endoparasitic nematodes have been increased recently. Although numerous species have been isolated from cysts and females, only a few species have been determined as parasites of nematodes. The most common species encountered in females and eggs are similar, but the taxonomically diverse mycofloras exist in different geographic locations. Although V. chlamydosporium is worldwide in distribution (Domsch, et al. ,1980) and has been reported as a major fungal parasite of H. avenae in Europe, the fungus was encountered with a low frequency in the egg masses of the Meloidogyne spp., and it was not found in cysts of H. glycines in subtropical Florida (Chen, et al. , 1996). The fungus appeared to be more adapted to temperate climates than to subtropical climates. Stirling and Kerry (1983) investigated fungi colonizing females of H. avenae in Australia. The most widely distributed parasite was V. chlamydosporium, but its abundance in soil was lower than the suppressive level as detected in a cereal fields in Europe. In contrast to V. chlamydosporium, N. vasinfecta is probably adapted to high temperatures of tropical or subtropical climates (Domsch, et al. , 1980). It was encountered at a high frequency in cysts of H. glycines in Florida (Chen, et al. ,1994), but with a low frequency in Illinois (Carris, et al. , 1989). Fusarium oxysporum has been encountered in cysts and females of nematodes at a high frequency at both high and low temperature climates. Nematophagous fungi have been recovered from a variety of habitats. Predacious fungi were isolated often from leaf mold, decaying wood, partly decayed plants, dung, living Bryophytes, and soil (Duddington, 1951). Gray and Baily (1985) surveyed the distribution of nematophagous fungi in a deciduous woodland. They found that nematophagous fungi were isolated throughout the soil profile and down to maximum of 35cm deep. Predators forming constricting rings, adhesive branches, and adhesive knobs were restricted to the upper litter and humus layers. The net-forming predators and endoparasites were isolated throughout the soil profile at all depths, although they were more abundant in the deeper mineral rich soil. In another survey, Gray (1983) found that nematophagous fungi were abundant in all habitats examined, although most widely in temporary agricultural pasture, coniferous leaf litter, and coastal vegetation. A number of species showed distinct habitat associations, in particular, A. robusta, A. musiformis and D. cionopaga. Most egg -parasitic fungi were isolated from females, eggs, egg masses and cysts of Hetroderidae. Egg- parasitic fungi can be found in various habitats. The fungal species isolated from cysts collected from pastures (Hay and Skip, 1993) were similar to those isolated from cysts collected from cultivated agricultural soils. Cysts and egg masses of the sedentary nematodes may provide unique niches to the egg- parasitic fungi. The eggs of these nematodes are aggregated. When these cysts and egg masses are exposed to soil, they will be vulnerable to the attack by fungi. The substrates of the cysts and egg masses may provide important nutrients for the fungi before the fungi parasitize the eggs. Migratory and free-living nematodes deposit eggs separately, making the observation of their egg - parasitic fungi difficult. Rhopalomyces elegans , one of the earliest studied parasites of eggs of many nematodes, is worldwide in distribution and can be found in soil, rotting plant debris, organic matters, and animal dung. 2.2.2.Nutritional adaptation of nematophagous fungi:- Nematophagous fungi are capable of using nematodes as the only or a part of their nutrient source. This ability may have developed over a long period of time in the evolution process. Limited attempts have been made to understand the evolution process of nematophagous fungi. Cooke (1962) hypothesized that competition stimulates the predatory activity of predacious fungi as an adaptation to overcome their low competitive saprophytic ability. This hypothesis was supported by some observations (Cooke,1963; Quinn,,1987). It appears that the development of predacious efficiency has been accompanied by the tendency to lose characters such as rapid growth rate and good competitive saprophytic ability that are associated with an efficient saprophytic existence in the soil (Cooke, 1963). The positive correlation between ability of nematophagous fungi to destroy nematodes and attraction of the fungi to nematodes also indicates the evolution toward dependence of the fungi on nematodes for nutrients. Belder and Jansan (1994) observed that formation of trapping devices was less temperature-and nutrient-dependent in simple adhesive hyphae than in complex adhesive network. Barron (1992) argued that many nematophagous fungi evolved from lignolytic and cellulolytic fungi. The evolutionary process is a response of these fungi to nutrient divergence in N-limiting habitats. This arguement was based mainly on a series of observations by Cooke (1962, 1963). Although the teleomorphs of most predacious Hyphomycetes are not known, some predacious fungi are members of the gilled fungi (Saikawa and Wadw,1986). Nutritional adaptation may also have evolved in opportunistic fungi associated with females, cysts, and eggs of sedentary nematodes. Even though a wide range of taxonomically diverse mycofloras have been encountered in females, cysts, egg masses, and eggs, the most common fungi are limited to a few genera, including Verticillium, Paecilomyces, Exophiala, Fusarium, Gliocladium and Phoma. Only a few fungal species have been associated with more than one species of nematodes, and the common fungal species isolated from cysts collected from different geographic locations and similar, suggesting that a degree of specialization may be a prerequisite for successful colonization of the ecological niches represented by eggs, females, or cysts of hetroderid nematodes (Rodriguez- Kabana and Morgan-Jones,1988). Many of these fungi are root colonizers and some are parasites of plants. Pyrenochaeta terrestris was commonly encountered in cysts of H. glycines and was pathogenic to the nematode eggs and soybean plants (Chen, et al. , 1996). Probably, the fungus was evolved from a plant- parasitic form to an organism capable of using both plants and nematodes as nutrient sources. Some non-phytopathogenic isolates of F. oxysporum are egg parasites of cyst and root-knot nematodes. Chen et al. (1996) compared the mycoflora of cysts of the soybean cyst nematode, H. glycines, with the mycoflora of egg masses of root-knot nematodes Meloidogyne spp. , in two adjacent fields on the same farm with similar soil texture and climate conditions. The similarity index for fungi colonizing Meloidogyne egg masses and H. glycines cysts was low (0.10- 0.18). In contrast, the average similarity index between different sample dates for fungi in brown cysts of H. glycines was 0.54, and the average similarity index between locations and sampling dates for fungi in cysts of H. glycines collected from different locations in the southeastern USA at different times was 0.48 (Chen et al. , 1994). This suggested that the mycoflora in egg masses of Meloidogyne species differed from that in the brown cysts of H. glycines. Although some fungi encountered in the cysts of H. glycines were also found in the egg masses of the Meloidogyne spp. , the frequencies of most fungi in in the egg masses differed from that in the cysts of H. glycines. Paecilomyces lilacinus colonized only 1 of 1711 brown cysts of H. glycines (Chen, et al. , 1994) that was compared with 26% of the egg masses of Meloidogyne spp. colonized by this fungus. Probably, the Floridian strain of P. lilacinus is more adapted to root-knot nematodes than to cyst nematodes. Mycofloras in cyst of H. glycines were compared among 45 fields in Minnesota (Chen and Chen, 2002). The Mycofloras in the cysts diversified, and no single highly pathogenic fungus dominated. 2.2.3. Interaction of nematophagous fungi with host nematodes:- Plant-parasitic nematodes spend a part of their life in soil and are subjected to attack by fungal antagonists. Nematode species and their density are a major factor influencing types and abundance of their predators or parasites. Migratory nematodes may be more vulnerable to attack by predacious fungi than the sedentary endoparasitic nematodes. Egg- parasitic or female-parasitic fungi may be more effective in regulating nematode population of sedentary endoparasitic nematodes, which deposit eggs in egg sac or retain the eggs within the female body, than the migratory nematodes, which deposit eggs singly. Egg-parasitic fungi may be more efficient in control of root-knot nematodes forming small galls than those forming large galls. Linford et al. (1937) demonstrated that addition of organic to soil resulted in marked changes in the indigenous nematode populations. Soil amendments increased free-living nematodes and decreased plant- parasitic nematodes (Gray,1987). It was suggested that the organic supplement, which led to increase in nematode density, also resulted in increased nematophagous activity of fungi in the soil. The enhanced activity of the nematophagous species present in the soil would then stabilize the rate of increase of the soil nematodes, and eventually reduce their population densities, including those of plant-parasitic nematodes (Gray, 1987). This hypothesis has been used as the basis for a number of experiments on the biological control of plant-parasitic nematodes in soil. Cookes (1962a, 1962b,1968) series of experiment suggested that in nature a simple labile equilibrium does not exist. Recent work indicates equilibria do exist between nematophagous fungi and soil nematodes (Jaffee, 1993; Gray, 1987). The dependency on nematode density and response to soil organic amendments vary among the predacious fungi (Cooke, 1963). Species that form either a branch, knob, or constricting ring are sensitive in forming traps in soil amended with organic matter, whereas net-forming fungi are not (Gray, 1987). The mode of action of organic amendments against nematodes consists of more than the direct effects on nematophagous fungi. Organic amendments can improve soil structure and soil fertility, alter the level of plant resistance, release toxic compounds, and stimulate antagonistic microorganisms (Stirling, 1991). All of these changes by organic amendments may affect nematodes dramatically. While the population of predacious fungi is related more to the nutritional value of the organic matter in soil than to the number of nematodes present, the population of obligate parasitic fungi is generally dependent on the density of host nematodes. The relationship between H. rhossiliensis and host nematodes is a good example. Jaffee et al. (1989) observed a spatial density-dependent parasitism of M. xenoplax by H. rhossiliensis in peach orchards in California. In a laboratory assay, the disease dynamics of H. schachtii exhibited temporal density-dependent parasitism (Jaffee, et al. 1992). The parasitism increased to nearly 100% with repeated addition of many hosts and declined to nearly 0% unless a minimum number of hosts (the host threshold density) was supplied. The decline of cereal cyst nematode in Europe was also a phenomenon of density-dependent parasitism of nematodes by fungi. In the first two years of monoculture, the cereal cyst nematode increased and reduced crop yield. With continuous monoculture, the nematode decline to less than damaging levels due to the increase of parasitism of the nematode by fungi, mainly N. gynophila and V. chlamydosporium.N. gynophila is an obligate parasite and its density of host nematodes (Perry 1978). Facultative-parasitic fungi associated with eggs, sedentary females, and cysts are mainly influenced by environmental factors and independent, or weakly dependent on nematode density (Chen and Chen, 2002). Verticillium chlamydosporium is a facultative parasite, and its density may be less dependent on the nematode populations than the obligate parasite N. gynophila. In a greenhouse study, however, V. chlamydosporium density increased with increasing nematode density (Bourne and Kerry, 1999). Presumably, the ability to parasitize nematode females and eggs gives the fungus a competitive advantage in soil. 2.2.4. Interaction of fungal antagonists of nematodes with other soil organisms:- Soil organisms represent almost all kinds of animals, fungi and bacteria. The interaction among the soil organisms is complicated but may be critical in determining the effectiveness of suppression of nematodes by their antagonists. The biggest problem in biological control of nematodes using nematophagous fungi is probably the difficulty of overcoming the competition from other soil organisms, especially in the rhizosphere, is useful in exploiting potential biological control agents, but limited studies have been done in this area. Some fungi are highly pathogenic to nematodes in the laboratory, but poor soil competitors. A. dactyloids is a predacious fungus forming constricting rings and also is able to colonize eggs of H. glycines (Chen, et al., 1996b). The fungus is ubiquitous in distribution but a poor competitor in soil. The fungus colonized a large portion of cyst and eggs, and reduced nematode populations. The fungus, however, was not found in cysts and eggs in untreated natural field soil (Chen, 1994). Similarly, H. rhossiliensis can grow and sporulate well in artificial medium, but the fungus seems to be a poor competitor in natural field soil (Jaffee and Zehr, 1985).
Some soil microspore animals may feed on fungi. Jaffee et al. (1997) observed enchytraeid worms fed on H. rhossiliensis. M. gephyropagum, A. thaumasia and A. hapotryla, and reduced the fungal population densities. In a later experiment, however, exclusion of enchytraeid, collembolans, and mite did not affect the population of H. rhossiliensis and M. gephyropagum (Jaffee, 1999). H. rhossiliensis was quite sensitive to biotic inhibition when formulated as palletized hyphae but insensitive when formulated as parasitized nematodes (Jaffee, 2000). In contrast, A. hapotryla was more sensitive to biotic inhibition when added to soil as fungus-colonized nematodes than as palletized hyphae. As hypothesized by Linford et al. (1937), soil bacterial population may affect predators and parasites of nematodes. Bacteria provide food for free-living nematodes also serve as a food source for many predacious fungi. Bacteria, particularly Pseudomonas spp., are the most abundant microorganism in the rhizospore. Actinomycetes can comprise up to more than one third of the total microflora associated with plant roots (Stirling, 1991). These organisms frequently produce antibiotics and inhibit fungal growth. Fungi such as Fusarium and Pencillium, Trichoderma, Aspergillus, Mortierella, Pythum and some species of the Mucorales are commonly isolated from the rhizosphere and bulk soil. Most of these fungi may not be pathogenic to plant-parasitic nematodes. Mycorrhizal fungi are important component of the rhizosphere mycoflora. Some mycorrhizal fungi are also egg colonizers (Francl and Dropkin, 1985). Mycorrhizal fungi can either estimulate or inhibit nematophagous fungi in the rhizosphere. Because the egg-parasitic fungi are generally closely associated with plant roots, mycorrhizal fungi may have great effects on this group of nematophagous fungi. Their colonization of the rhizosphere may prevent colonization of roots by nematophagous fungi. Competition for nutrient between nematophagous fungi and saprophytic fungi may also occur in cysts and egg masses. Many fungi isolated from cysts and egg masses are saprophytic. While Hay and Skipp (1993) repoted that multi-species of fungi are commonly encountered in cysts of Hereroders trifolii collected from pasture soil in New Zealand, many studies (Carres, et al. 1989; Chen, et al., 1994; Gintis, et al., 1983; Morgan-Jones and Rodriguez-Kabana, 1985) suggested that cysts colonized by one fungus are not readily colonized by other fungi. Colonization of cysts by non-antagonistic, saprophytic fungi may inhibit egg-parasitic fungi and therefore protect nematode egg from being destroyed by the fungi (Chen, 2003). 2.2.5. Effect of Plants and Cropping System:- Crop species and cultivars influence microflora and fauna in soil, especially in the rhizosphere. A number of factors affect the root exudates; the principal factors are the species and developmental stage of the plants. There is some evidence that fungal growth is affected by root exudates (Vancura, 1988). Only limited studies, however, have been done to investigate the effect of plant species on the nematophagous fungi (Bourne and Kerry 1999, 2000). Plant species differed in their ability to support growth of V. chlamydosporium in their roots or rhizosphere at different nematode densities; fungal density on the roots of some species than others (Bourne and Kerry, 1999; Bourne, et al. , 1994). In nutrient poor sandy soil, the rhizosphere of tomato roots was able to support a larger population density of the egg- parasitic fungus, V. chlamydosporium, than the bulk soil (Bourne and Kerry, 2000). Similarly, the population densities of the predacious fungi, A. dactyloides, A. superba, and Monacrosporium ellipsosporu, were greater in tomato rhizosphere than in root-free soil (Person and Jansson, 1999). The ability of colonization of roots is essential for effective control of root-knot nematodes by fungi. The locations and size of egg masses of root-knot nematodes vary among the species and their hosts. Egg masses on root surfaces may be more vulnerable to attack by egg-parasitic fungi, while egg masses formed inside roots may be able to escape the fungal attack. Dactylella oviparasitic suppressed populations of M. incognita on peach tree in a California orched but not on grape plants in adjacent vineyards. A greenhouse experiment showed that the nematode produced smaller egg masses on peach than on grape; D. oviparasitic parasitized most egg formed on peach and only about 50% of egg on grape (Stirling and Mankau, 1979; Stirling, et al. 1979). It was suggested that the size of egg masses determines of root-knot nematode by D. oviparasitic. A limited number of studies have been reported on the effects of crop sequences on nematophagous fungi. Steudel et al.(1990) studies the effect of sugar beet- cereals-green manure rotation on the H. schachtii population density and the frequency of its fungal parasites of eggs. The highest level of parasitism of the nematode by fungi was observed after years with sugar beet. In the interim, parasitism deceased to about 10%. No difference of fungal parasitism was observed among plots planted to oil radish, white mustard, "Pegletta,"or fallow. A high nematode multiplication on susceptible cach crop promoted antagonistic fungi; consequently the nematode population density decreased more quickly following sugar beet. Chen and Reese (1999) demonstrated the effect of crop sequence of H. glycines by the endoparasite H. rhossiliensis. The percentage of H. glycines J2 parasitized by the fungus increased in the first years of soybean after 5 years of corn. Fungal parasitism was similar in plots of second through 5th years of soybean after 5 years of corn and in plots of soybean monoculture.When corn was planted, parasitism of H. glycines J2 decreased. The effect of crop sequence on the fungal parasitism of H. glycines J2 may be attributed to a density-dependent relationship between the parasite and its host nematode.Bernard et al, (1997) investigated the incidence of fungi invading females, cysts, and egg of H. glycines in various cropping-tillage plots. No significant difference in percentage of cysts or female colonized by fungi was observed among six cropping-tillage. The diversity of egg-parasitic fungi was similar among the treatments. Although the percentage of egg parasitized by total fungi did not differ among the cropping-tillage treatments, P. lilacnus and V. chlamydosporium occurred more frequently in the moldboard plow treatment than in any treatment. 2.3.Effects of soil a biotic factors:- Soil abiotic variables, such as soil texture, temperature, moisture, nutrients organic matter, and pH may determine microflora and fauna in the soil (Gray, 1987; Kerry, 1993). These soil factors affect activities of fungal antagonists of nematodes directly or indirectly. It may be impossible to use microbial agents efficiently for control of nematodes without understanding how the agents interact with soil abiotic factors. 2.3.1. Soil temperature:- Optimum temperature for growth and development differs among nematophagous fungi. Predacious fungi may require a low temperature for their growth and predacious activities (Belder and Jansen, 1994b). Isolates of M. ellipsosporum from Antarctica adapted to a lower temperature than did isolates from UK (Gray, 1982). The endoparasitic fungus H. rhossiliensis, infects J2 of H. schachtii fastest at 25ºC to 30ºC (Tedford, et al. , 1995). The optimum temperature for control of root-knot nematodes by V. chlamydosporium is 25ºC (De Leij, et al. , 1992b). When the temperature exceeded 25ºC, the percentage of eggs of the nematodes parasitized by the fungus decreased; at these temperatures the nematodes developed fast and hatched before they were colonized by the fungus. Difference in temperature requirement exists among isolates of V. chlamydosporium (Kerry, 1981). Nematophthora infected more females of H. schachtii at 10ºC and 15ºC than at 20ºC (Kerry, 1984). D. oviparasitica grew most rapidly at 24ºC than at 27ºC (Stirling, 1979). In contrast, C. anguillulae infected more nematodes at 24ºC – 28ºC than at 12ºC (Sayre and Keeley, 1969). 2.3.2. Soil moisture:- Nematodes are aquatic animals and need a film of water in soil for microspores moving and living. Nematodes are most active when soil moisture is at or close to field capacity (Jones, 1975). At low soil water potentials from -0.1 bar to - 0.25 nematodes may be immobilized. Mobility of nematodes determines the chance of contacting their natural animals, especially predacious fungi and some other fungi that do not actively move in soil. Movement of soil water may disseminate fungal spores. Gray (1985) found that species producing adhesive networks were isolated more frequently from soils with low moisture. In contrast, ring-forming species were isolated significantly more frequently from soil with relatively high moisture and organic matter contents. Fungi can grow and develop in relatively dry soils; their growth is not restricted when the soil water potential is above -30 bar. Nematophagous fungi that produce motile zoospores are more active in soil with higher water potentials. Parasitism of H. avenae by N. gynophila and C. auxiliaries was higher in dry seasons (Kerry, et al. ,1982b; Stirling and Kerry, 1983). Another effect of moisture on fungi is related to soil aeration. Establishment of V. chlamydosporium was better, and control of M. incognita with the fungus was greater in well aerated soil than in soil that was less aerated (Kerry, et al. , 1993). Similarly, infection of H. rhossiliensis was higher at low water potentials than at high water potentials (Tedford, et al. , 1992). 2.3.3. Soil Texture:- Soil texture refers to the relative proportion of particles of different sizes. The size of soil particles determines the total surface, with which soil chemical compounds, microorganisms, and plant roots interact. Light soils are generally favorable to large populations of nematodes. The size of soil micropores influences both dispersal of nematophagous fungi and the movement of nematodes. Soil texture affects the proliferation and survival of fungi in soil. Tedford et al. (1992) reported that transmission of H. rhossiliensis to H. schachtii was greatest in loamy sand, intermediate in loam, and lowest in sand. Water drainage and aeration are generally better in sandy soils than in heavy clay soils; hence a sandy soil may favor fungal growth if adequate nutrients are provided. Also nematode movement may be better in soil with better drainage and aeration. Therefore, it may be expected that a light sandy soils have low organic matter contents, and consequently saprophytic activity of fungi may be low. Combination of fungal agents and organic amendments may be a good practice for control of nematodes in a light soil with some facultative parasites. The bulk soil is a major barrier for biological control agents to reach target nematodes. Generally, a large dosage is needed to ensure effectiveness of an immediate control of nematodes. 2.3.4. Effect of soil pH, nutrients, Organic matter, and Agricultural chemicals:- Soil chemical characteristics are important factors influencing nematophagous fungi. Different fungi may have different requirements of soil pH. While the ring- and knob-forming species are associated with soils of a low pH, species with unmodified hyphae were isolated significantly more frequently from soils with a higher pH. Conidium-forming endoparasites were isolated from samples with relatively low soil pH (Gray,1987). Optimal pH for P. lilacinus sporulation occurred at pH 4 – 6 (Villanueva and Davide, 1984). Activity of palletized H. rhossiliensis was negatively correlated with soil pH (Jaffee and Zasoki, 2001). Maximum activity occurred at pH 4.5, and activity gradually declined to near zero as the pH increased to 6.5 and rapidly declined to near zero as the pH dropped below 4.0. It is concluded that low soil pH suppresses soil organisms that otherwise interfere with growth of H. rhossiliensis from alginate pellets.
Gray (1987) studied the effects of soil nutrients N,P, and K on predacious fungi and indoparasitic fungi. Endoparasites with adhesive conidia were independent of soil nutrients, while those species with ingested conidia were isolated from soil with high concentrations of nutrients. Knob-forming predators, which rely on their ability to produce traps spontaneously, were isolated from soils with low concentration of nutrients, while those species with constricting rings were isolated from richer soils, which contain greater densities of nematodes Net-forming species were largely independent of soil fertility, although generally they were isolated from soils with limited nutrients, specially low K. Gray (1985) studied the effects of organic matter on endoparasitic fungi and predacious fungi of vermiforms nematodes. Those endoparasites producing conidia were significantly associated with soils with high organic matter. Ring-forming species were isolated more frequently from soil with relatively high organic matter contents, while species producing adhesive networks were isolated more frequently from soils with low organic matter contents. Predacious fungi may increase in response to the decomposition of organic matter. Timm et al. (2001) demonstrated that frequencies and population densities of most species of predacious fungi were similar in conventionally and organically managed plots. In contrast, parasitism of M. xenoplax by H. rhossiliensis was decreased by addition of organic matter (Jaffee et al. ,1994). Recently, studies of the effects of soil amendments on egg and female parasites have increased. The effect of organic matter on egg- parasitic fungi may depend on the type of organic matter and the soil biological communities and environmental conditions. Pyrowolakis et al. (1999) demonstrated that green manure increased egg- parasitic activities of fungi on H. schachtii in soil from a man gold-monoculture field and reduced the activities in soil from a field in sugar beet-wheat rotation. Straw fertilizer favored more fungal parasitism of H. schachtii eggs than manure (Sosnowska and Banaszak, 2000). Some studies have demonstrated that amendments of chitin or collagen into soil suppressed plant-parasitic nematode populations. Some studies, however, disproved the idea that organic amendments stimulated the fungal parasitism of eggs or females (Mankau, 1961; Rodriguez-Kabana et al. ,1984a). Effect of agricultural chemicals on nematophagous fungus is poorly understood. Crump and Kerry (1986) tested the effects of 25 chemicals on the growth of V. chlamydosporium. Of the nematicides tested, oxamyl was the only one that decreased growth of C. destructans and V. chlamydosporium. The insecticides and herbicides tested had little effect on the fungal growth. Fungicides used to control plant diseases may also suppress fungal parasites of nematodes but it has not been well studied. 2.4. Biological Control 0f Nematodes by Fungal Antagonists:- The action of antagonists in maintaining nematode population density at a lower average than would occur in their absence can be called biological control of nematodes. Nematode antagonists have been observed in a wide range of organisms including fungi, bacteria, viruses, richettseae, plants protozoans, mites, insects and nematodes. Among these, fungal antagonists have been most important in regulating nematode populations in soil. Fungal antagonists of nematodes have been studied since the first observation of the nematophagous habit of the fungus, H. anguillulae, by Lohde in 1847. Linfords (Linford, 1937; Linford, et al., 1938) effort to use predacious fungi to control plant parasitic nematodes stimulated interest in the nematode - predacious fungi. Early research done in France, the USA, England. The interest in biological control occurred in the mid- 1970s. This resulted from both the continuing environmental problems associated with the use of nematicides (Kerry,1993; Stirling,1991) and demonstrations of suppressions of nematodes by fungal parasites. Some efforts have been made to market biological control agents (Cayrol, et al. 1978; Liu, et al., 1996; Timm, 1987; Warrior, et al., 1999) for nematode management, but the products generally have not been accepted or they used only at small scale. Recently, more and more evidence showed that biological control of plant –parasitic nematodes with fungal antagonistic is promising. Biological control of plant- parasitic nematodes has been at a crucial stage where successful examples of using nematode antagonists in management are needed to warrant continuous supports from public and industries. 2.5. Types of fungal antagonists of nematodes:- Fungal antagonists of nematodes are those fungi, including nematophagous and non-nematophagous fungi that have some adverse effect on nematodes. Numerous fungi have been isolated from nematodes. Cooke and Godfrey (1964) published a key that included 97 predacious and endoparasitic fungal species attacking vermiform nematodes. Crump (1991) listed 129 species of fungi isolated from beet, cereal, and potato cyst nematodes. Rodriguez- Kabana and Morgan-Jones (1988) summarized fungal species isolated from root-knot and cyst nematodes. Many more fungal species have been reported recently from nematodes throughout the world. Li et al.(2ooo) did an extensive taxonomic review of nematophagous fungi. Nematophagous fungi use nematodes for nutrition. In early literature, nematophagous fungi are generally referred to as predacious (trapping) and indoparasitic fungi. Barron (1977) provided a detailed description of the predacious and endoparasitic fungi of vermiform nematodes. After more knowledge of fungi associated with eggs, females, and cysts sedentary endoparasitic nematodes, such as Heteroderidae was accumulated, a third group of nematophagous fungi was proposed: Opportunistic fungi (Morgan-Jones and Rodriguez- Kabana, 1987). According to the mode of action, the fungal antagonists of nematode can be grouped into: (1) predacious fungi; (2) endoparasites of vermiform nematodes; (3) parasites of sedentary females and eggs; (4) fungi producing antibiotic substances; and (5) vesicular-arbuscular mycorrhizal (VAM) fungi. 2.5.1. Predacious fungi:- Predacious organisms capture, kill, and then consume their prey. Like some omnivorous plants, predacious fungi have evolved special devices for capturing animals such as vermiform nematodes. These devices are adhesive hyphae, adhesive branches, adhesive nets, adhesive knobs, non- constricting rings, constricting rings and stephanocysts (Barron, 1977; Liou and Tzean, 1992). However, killing of nematodes by some predacious fungi may be a slow process and fungi may undergo parasitism for along period. These fungi are considered parasites of nematodes as well. 2.5.1.1.Fungi with adhesive hyphae:- Adhesive hyphae are usually produced by lower fungi most of which are in the genera Stylopage and Cystopage, Zygomycetes. Because they have no septa, they cannot produce complex devices for capturing nematodes (Barron,1977). However some septate fungi in Deuteromycetes, such as A. botryospora, D. psychrophila. And A. superba, also can capture nematodes with adhesive hyphae under certain conditions. These fungi produce adhesive materials that are deposited on some points of hyphal surfaces. When a nematode touches these points, it is captured. The hyphae produce appressoria that penetrate through the nematode body. After consuming all of the nematode contents, the fungus draws all plasma back outside of the nematode body for the development of sporangia and spores. 2.5.1.2. Fungi with adhesive branches:- Adhesive branches are produced by Deuteromycetes and lower fungi such as Stylopage. The behavior of these fungi is similar to that of adhesive hyphae. Adhesive branches are usually made up of one to three cells and normally anastomose to form simple adhesive hoops or two-dimensional networks. A thin film of adhesive materials is secreted over the entire surfaces of branch. The trap is elevated above the substrate. Nematodes that become attached to branches are held fast, and they are quickly penetrated and consumed. The adhesive branches are probably primitive traps from which complex organs of capture have evolved (Barron 1977). 2.5.1.3. Fungi with adhesive nets:- Adhessive nets are common trapping devices of predacious fungi and usually found in the Deuteromycetes. These fungi have been studied intensively to determine there mechanism of infection of nematodes and there efficacy in reducing nematode densities. Nordbring-Hertz (1972) demonstrated the presence of an adhesive substance coating the surface of capture organs. The adhesive nets are effective; Once nematode is captured, the prey is held fast. If the nematode struggle, it becomes attached to another net and may become attached to several nets or branches (Barron, 1977; Gray,1988). Large nematode as well as small nematodes can be captured by this device. Once a nematode is captured, a penetration hypha enters the nematode and then swells to form an infection bulb. From this structure assimilative hyphae developed 2.5.1.4. Fungi with adhesive knops:- Adhesive knops are morphologically distinct cells, and they are considered to be highly specialized trapping devices. These knops are covered by a thin layer of adhesive materials and are either sessile or borne at the apex of of a short non-adhessive stalk. When a nematode touches the knops, a flattened mass of adhessive is produced at the point off attachment, forming a thick pad. This increase the area of attachment many folds, insuring that the nematode is firmly held (Gray, 1988). Adhessive knops contain numerous electron-dense spherical bodies close to the part of the cell wall where is likely to come into contact with the nematode (Wimble and Young, 1984). Adhesive knops are normally quite closely spaced a long hyphae, and when a nematode is caught it is quite normal for it to become attached to several other knops as it struggles to free it self, making escape impossible (Gray,1988). In the struggle to escape, the nematode usually breaks the knob off and carries the knob on its body surface. The knob then penetrates the host and the nematode is parasitized. The adhesive knobs are found in Deuteromycetes, such as D. candida, produce a globose infection bulb after penetration. From this bulb, assimilative hyphae arise to colonize and digest the host contents. Basidiomycetes, such as Nematoctonus sp. , do not form an infection bulb after penetration. This group of fungi is unique in forming the clamp connections on the secondary hyphae. 2.5.1.5 Fungi with non-constricting rings :- Non-constricting rings are produced by erect, lateral branches arising from the prostrate septate hyphae of Deuteromycetes, such as Dactylaria. Fungi that produce non-constricting rings, as exemplified by D. candida and D. lysipaga, often produce adhesive knobs as well (Drechsler,1937). Knobs and rings are commonly found alternating on the same hyphal strand (Gray,1988). Also, an adhesive layer, similar to those associated with adhesive nets and adhesive knob traps, has been observed on the inside of the ring (Dowsett and Ried,1977). The ring is composed of three cells. When a nematode enters the ring and if its diameter is only a little larger than the inner diameter of the ring, the ring will hold the nematode. The connection between the ring and stack is very delicate and the nematode usually will break off the ring in its struggle to escape. One nematode may carry several rings around its body. Finally the fungus penetrate the nematdode body wall and consumes the nematode. 2.5. 1.6 Fungi with constricting rings :- The most sophisticated device for predacious fungi is the constricting ring formed by species of Dactylaria, Dactylella, Arthrobotrys and a few other Deuteromycetes. The constricting rings, which also consist of three cells, similar to non-constricting rings in morphology. However, unlike the non- constricting ring, the constricting ring is borne on a strong stalk that is not easily broken. The size of the ring varies between and within species, but generally falls with a range of 20 µm to 40 µm (Gray,1988). When the constricting ring is stimulated, the three cells expand rapidly inward. If a nematode moves into the ring, the inner walls of the ring cells will be triggered to close, and the nematode will be pinched and have no chance to escape. The mechanisms involved inclosure of the rings, however, are debatable. Both mechanical and hot water, but not chemical stimulation have triggered the swelling of the constricting rings (Couch, 1937; Muller,1958;). Couch (1937) believed that the closure was affected by swelling of a colloidal substance. The swelling may be due to rearrangement of molecules of water and colloidal material already in the cell, or it might be that additional water is imbibed from the stalk and thread cells. Muller (1958) argued that colloidal material could not account for ring closure but rather stimulation of the inner walls of the three cells changed the permeability of the cell membranes and the cells absorbed water from the surrounding medium, causing the ring to close.
2.5. 1.7. Stephanocysts:-
Stephanocysts are unique reproductive structures, apparently a kind of conidia or spores, present in Basidiomycetes, especially in the genus Hyphoderma (Liou and Tzean.1992). A typical stephanocyst consists of a cup-like basal cell and a terminal globose cell. At the juncture of the two cells, a row of spines surrounds the circumference. Liou and Tzee-an(1992) observed that the stephanocysts also function as nematode-trapping devices. The two-celled stephanocysts are adhesive, and can attach to passing nematodes while attached to or detached from the hyphae or short stalks. Once a nematode is captured, the fungus forms infective peg to penetrate, and then consumes the prey nematodes.
2.5.2. Endoparasites of Vermiform nematodes:-
Endoparasitic fungi are different from the predacious fungi in that they have no special trapping devices. Most endoparasitic fungi of vermiform nematodes are obligate parasites or have limited saprophytic ability. They have no extensive hyphal development outside the body of nematodes. Some fungi attacking vermiform nematodes, however, are facultative parasites and can undergo saprophytic activity without nematodes.
2.5.2.1. Obligate endoparasites of vermiform nematodes :-
The obligate endoparasitic fungi that attack vermiform nematode can be found in lower and higher fungi. They can be placed into four groups based on their mechanism of infection: (1) encysting species; (2) species forming adhesive conidia; (3) species with conidia that may be ingested (Barron, 1977; Gray, 1988); and (4) species with gone cells (Barron, 1987; Rob and Barron, 1982). 2.5.2.1.1. Encysting species:- Encysting species that produce zoospores belong to Chytridiomycetes and Oomycetes. The zoospores can swarm for a short time, and if they reach their host nematode they encyst on nematode cuticles. Catenaria spp., which belong to chytridiomycetes, are the most common endoparasitic fungi encountered on vermiform nematodes. Their zoospores have one posterior whiplash flagellum, and sporangia are formed in chains within the nematode body. Myzocytium spp. (Oomycetes), which also can parasitize nematodes, are different from Catenaria spp. In that they produce oospores and zoospores with two flagella, one tinsel and the other whiplash. 2.5.2.1.2. Species forming adhesive conidia:- The endoparasitic fungi forming adhesive conidia are found in Zygomycetes, Bassidiomycetes, and Deuteromycetes. Species of Meristacrum (Zygomycetes) form conidia that are forcibly discharged at their maturity. If the primary conidia fail to contact a nematode, they may germinate and produce a secondary conidium. Both primary and secondary conidia have a layer of sticky material on their surfaces. The endoparasitic Basidiomycetes such as some species of Nematoctonus also infect nematodes using adhesive conidia, but the conidia formed by species of Nematoctonus are similar to the adhesive knobs formed on hyphae by the predacious species. Most endoparasitic fungi forming adhesive conidia were found in Deuteromycetes such as many well-known species in the genera Verticillium, Cephalosporium, Acrostolagmus and Hirsutella. The infective conidia produced by these fungi either have adhesive mucilage in parts of the surface of the conidia or have mucous sheath enveloping conidium. Although the saprophytic abilities of these fungi in natural soil are not well studied, most of these fungi can be extensively cultured on artificial media and produce infective conidia. Therefore they may be considered facultative endoparasites. H. rhossiliensis, however, behaves as an obligate endoparasite in nature soil because it will go extinct without supplying a minimum number of host nematodes (Jaffee and Zehr,1985). 2.5.2.1.3. Species with conidia that may be ingested:- Some species of endoparasites have developed morphologically adapted conidia, which, when ingested by a nematode, become lodged in either its buccal cavity or esophagus. The species belong almost exclusively to the genus Harposporium (Deuteromycetes). The conidia are crescent-shaped, heliocoid, or irregular-shaped, and they are well adapted to infection. These fungi cannot infect plant parasitic nematodes because the clumen of the stylet is too small to ingest the conidia. 2.5.2.1.4. Species with gun cell Species of Haptoglossa (Oomycetes) have evolved a special infective cell called (gun) cell to attack nematode or rotifer hosts (Barron,1987; Beakes and Glocking,1998; Robb and Barron, 1982). The biflagellate zoospores are discharged from zoosporangium, swim a short distance, and encyst on substratum within a few minutes, After 2-3 h of quiescence, the encysted spore undergoes synchronous germination, initially producing a narrow germ tube. The germ tube soon begins to swell and within an hourvor so forms the ovoid to slightly tapering gun cell, which is delimited from the early empty cyst by the thin cross wall. The infective cell takes about another 2-3 h to become mature, and by then the original cyst often appears collapsed and the gun cell has acquired a prominent apical beak, often held at an acute angle to the main cell body (Beakes and Glockling, 1998). The gun cell often anchors on substrate in the manner of a cannon. Once a cannon-like gun cell, it triggers the cell to fire a (missile) through the host cuticle and then serve as a hypodermic conduit through which the parasitic cytoplasm is injected into the host (Breaks and Glocking,1998). 2.5.2.2. Facultative parasites of vermiform nematodes:- Whereas the saprophytic ability of many fungal endoparasites of vermiform nematodes in nature is unclear, a few fungi such as the species C. anguillulae can grow saprophytically on dead or injured nematodes (Sayre and Keeley,1969). Many fungi of Deuteromycetes that produce adhesive probably appropriate to group these fungi with facultative parasites. A recently described fungus Esteya vermicola that was isolated from the pinewood nematode Bursaphelenchus xylophilus produce infective, adhesive conidia when cultured on poor nutrition media or grown from parasitic nematode, and produced non-infective, non-adhesive conidia when cultured on enriched media (Liou, et al. ,1999). Like predacious fungi, E. vermicola might have adapted to both saprophytic and parasitic growth. Some predacious fungi, such as those with adhesive knobs, act very similarly to these endoparasitic fungi that form adhesive conidia. Consequently, the predacious fungi with adhesive knobs can be considered facultative endoparasites of vermiform nematodes. Many fungi, such as V. chlamydosporium, that colonize cysts or egg masses also can attack second- stage juveniles within the cysts or egg masses. Although the mode of infection of juveniles in cysts or egg masses is poorly understood, these fungi are probably facultative parasites of vermiform nematodes. 2.5.2.3. Parasites of sedentary females and eggs Sedentary nematode females, cysts, eggs and egg masses are also subjected to the attack by fungi. Unlike motile vermiform nematodes they may actively move toward and contact predacious or endoparasitic fungi, sedentary stages of nematodes may have no chance to contact parasitic fungi unless the fungi have some mechanism to reach the nematodes Fungi attaching sedentary nematodes diversify biologically and ecologically. While a few of them are obligate parasites, most fungi in this group can live in soil as saprophytes. Mechanisms of attacking nematodes differ between obligate parasites and facultative parasites. 2.5.2.3.1. Obligate parasites of sedentary females and eggs Obligate parasites of sedentary females and eggs cannot develop mycelium in soil. Therefore, these fungi must have a motile or disseminative stage in order to reach the target nematode. Three species, C. auxiliaries, Nematophthora gynophila, and an undescribed lagenidiaceous fungus, have been reported on cysts nematodes (Kerry and Crump, 1980). All of these fungi produce thick-walled resting spores that remain in soil from one season to the next. Zoospores are probably the infective stage (Stirling,1991; Tribe,1977). The zoospores move toward nematode female, and when they contact the target nematode, they penetrate the body wall and assimilate the nematode body contents. C. auxiliaries and N. gynophila also attack nematode eggs within the female body (Tribe, 1977). 2.5.2.3.2. Facultative parasites of eggs and sedentary females:- Numerous fungi have been isolated from eggs, sedentary females, egg masses, or cysts of Heteroderidae. Some of them may not be parasitic species, while others are facultative parasites. The facultative parasitic fungi produce an expanding mycelium growth that enables them to reach the nematode targets of sessile stages. These fungi belong to taxonomic divergent groups of Chytridiomycetes, Basidiomycetes, and Deuteromycetes, but they are encountered more frequently in Deuteromycetes. The existence of mycofloras associated with eggs, egg masses, females, or cysts of nematodes belonging to the Heteroderidae has been known for more than a century. As early as 1877, kuhn reported a fungus Tarichium auxiliarum, that parasitized females of H. schachtii (Morgan-jones and Rodriguez-Kabana,1988). During The 1920s and 1930s a large number of cysts in several survey were examined in Europe for fungal parasites of the cyst nematodes, H. schachtii and H. avenae (Morgan-jones and Rodriguez- Kaban,1988), Recently, studies on the species and frequency of fungi associated with cyst and root-knot nematodes have increased. Rodriguez- Kabana and Morgan-Jones (1988) reviewed the fungi isolated up to 1988 from Heteroderidae collected from Australia, Europe, North America and South America. Since then, many more fungal species have been reported from these nematodes collected in various geographic locations. The fungal species most commonly isolated are limited to a few genera, indicating that there is a certain degree of specialization towards the nematodes. These fungi include Alternaria, Catenaria, Fusarium, Nematophthora, Paecilomyces, Penicillium, Phoma and Verticillium. Besides these fungal genera, Neocosmospora vasinfecta and Stagonospora heteroderae were frequently isolated from H. glycines in warm climatic conditions (Chen, et al., 1994). 2.5.2.4. Fungi producing antibiotic substances:- Many fungi isolated from cysts and egg masses are probably saprophytic. Their effects on nematode are not clear. Presumably, some of these fungi produce substances toxic to nematodes or their existence in egg masses or cysts inhibts or stimulates hatching of juveniles from eggs. Toxic effects of fungal culture filtrates on vermiform nematodes and eggs have been reported in a number of studies from several fungi such as species of the genera Paecilomyces, Verticillium, Fusarium, Aspergillus, Tricoderma, Myrothecium and Penicillium. Tricoderma species have high enzyme activities and it could be usefully utilized to control the disease in vivo or other wise to control the causal organismin plant debris buried in soil. This illustrates the importance of testing the ability of Trichoderma to survive, spread, infect and decrease populations of microorganism in the soil. Tricoderma sp. were reported to be highly effective in controlling soil-borne pathogens ( Budge and Whipps, 1991; Chet,1987; Chet et al. 1979; Cole and Zvenyika, 1988; Lewis and papavizas,1984). A few studies have been done to characterize toxic compounds produced by fungi. P.lilacinus releases chitinase and protease, which can cause undifferentiated M. hapla eggs to become deformed and vacuolate (Fitters, et al., 1992). Meyer et al.,(2000) reported that non- enzymic factors produced by T. virens (syn. Gliocladium virens) inhibited M. incognita egg hatch and 12 mobility. Toxins produced by Fusarium spp. Were tested on M. incognita and some were highly toxic to the nematode (Ciancio, et al.,1988). An antibiotic from Cylindrocarpon olidum was isolated, purified and characterized (Coosemans,1991). It showed good nematicidal activity and had low toxicity towards vertebrates. Purified extracts of Penicillium sp., P. anatolicum and Aspergillus niger showed high nematicidal activity at 100 ppm and 200ppm (Molina and Davide,z1986). Fungi that produce antibiotic substances may be common in soil. Many more soil fungi that are antagonistic to nematodes through the release of toxins, antibiotics or enzymes remain to be discovered. Nematode density has been correlated negatively with activities of chitinase, collagenase and proteinase of some soil microorganisms (Mian et al., 1982; Rodriguez- Kabana, et al.1989), including fungi such as Cunninghamella elegans (Galper, et al., 1991). Kloepper et al. (1991) observed that plants with properties antagonistic to plant-parasitic nematodes have rhizosphere microphlora distinct from those of host plants , and they also found that greater numbers of microorganisms in the rhizosphere of antagonistic plants were chitinolytic. Whether the mycofloras are involved in the antagonism of the plants to nematodes through the release of substance is worthy of study. 2.5.2.5. Vesicular-arbuscular Mycorrhizal The vesicular-arbuscular mycorrhizal (VAM) fungi are indomycorrhizal fungi that invade deeply into roots. All VAM fungi belong to the order Glomales (Zygomycetes). The symbiotic association is obligate for these fungi, and they have not been successfully cultured apart from their hosts. During the past two decades many studies on effects of VAM fungi on nematodes have been reported. The roles of VAM fungi in regulating nematode populations and their modes of action have not been educated fully. The response of nematodes to VAM fungi varies and may depend on the specific association, soil nutrient level and timing of the observation (Ingham,1988). Both antagonistic and beneficial effects of VAM on nematode populations have been reported. The VAM fungi may complete for nutrition and root space, modify root exudates, alter plant physiology, colonize nematode feeding sites, reduce the number of giant cells, or release nematoxin or antibiotics (Ingham,1988; Suresh, et al., 1985; Bagyaraj, et al., 1979). On the other hand, AVM fungi may improve plant growth and offset the yield loss normally caused by nematode parasitism, and increase food sources for nematodes thereby increase nematode populations. Francl and Dropkin (1985) reported that Glomus fasciculatum could parasitize H. glycines eggs but was not sufficient to effectively reduce nematode population density. As a group, VAM fungi cannot be placed in any group discussed above and thus it considered to be a unique type of fungal antagonists. More detailed reviews on interaction between VAM fungi and nematode populations have been provided by Ingham (1988). 2.6. Modes of infection of nematodes by fungi:- The knowledge of mode of infection of nematodes by fungi is important for using them as biological control agents. Most research on mode of infection of nematodes was conducted on predacious and endoparasitic fungi that attack vermiform nematodes. The relationships of A. oligospora or Drechmeria coniospora with nematode have been used as models for studying the recognition mechanism of fungal infection. Various aspects of modes of infection have been intensively (Dackman, et al., 1992; Dijksterhuis, et al., 1994; Nordbring-Hertz, 1988; Nordbring-Hertz, et al., 1981; Tunlid, et al., 1992). 2.6.1. Attraction Attraction between nematodes and nematophagous fungi was observed in many studies. The attraction of fungi to nematodes increased with increasing dependence of the fungi on nematodes for nutrients (Jansson and Nordbring-Hertz, 1979, 1980). Several factors, such as carbondioxide and various organic and inorganic substances, have been thought to be involved in nematode attraction (Gray,1988). A volatile or a small rapidly diffusing compound was continuously produced in non-spontaneous trap formers ( fungi insensitive in trap formation in response to organic matter, including net-forming species). Spontaneous trap formers (fungi sensitive in trap formation in response to organic matter, including branch-, knob-and ring- forming species) and endoparasites produced larger, or less volatile, slowly diffusing compounds, which attract nematodes (Gray, 1988; Jansson and Nordbring-Hertz, 1979). Sialic acid located on the head region of nematodes was found to be involved in chemotaxis of zoospores to nematodes (Jansson and Nordbring-Hertz, 1984). The motile zoospores of endoparasitic fungi are thought to be attracted to nematodes as a result of the chemical gradients form by exudates released from nematode body orifices. It is not clear if there is a chemotropic response of facultative fungal parasites of eggs or females towards their sedentary hosts. Zoospores of C. auxiliaris (Tribe, 1977) and N. gynophila (Kery,1974), however, are thought to move towards nematode females. 2.6.2. Attachment When nematodes come in contact with nematophagous fungi, some morphological changes and chemical interactions may occur. The binding sites vary among combinations between nematode and fungi (Durschner- pelz and Atkinson,1988). Three patterns of binding sites of nematode with fungi have been observed: (1) specifically to the head and tail; (2) all over the body; (3) very sparse or no binding (Jansson, et al.,1985). Zoospores usually attach near natural body openings. Molecular moieties are responsible for recognition between nematodes and nematophagous fungi (Tunlid, et al., 1992). Although it had been suggested previously that predacious fungi were not nematode-specific (Rosenzweig, et al.,1985), the results obtained by Gaspard and Mankau (1987) indicated that host – specificity does exist in the interaction between predacious fungi and nematodes. Binding of lectin carbohydrates is involved in recognition between predacious fungi and nematodes (Wharton and Murry, 1990). The qualitative and quantitative variation in carbohydrates on the surface of different nematodes may provide more opportunity for ecological specialization of endoparasitic fungi that use lectins in host recognition. Attachment and penetration of nematodes by fungi are two separate events. Blocking the binding sites by saccharides prevented penetration of Tricostrongylus colubriformis by A. oligospora, but had no effect on capture of the nematode; thus capture and penetration may be two distinct processes, with capture being less specific than penetration. Freeze-dried conidia of D. coniospora attached to Caenorhabditis elegans and Panagrellus redivivus as readily as did untreated conidia, but infection was significantly delayed (Zuckerman, et al., 1988). 2.6.3. Penetration Many light and electron microscopic studies have been published showing the penetration of nematode cuticle, mainly vermiform nematodes, by different nematophagous fungi (Jansson and Nordbring-Hertz, 1988) whether penetration of the cuticle is enzymatic or mechanical, or both, is not fully understood. Organelles such as endoplasmic reticulum in infective hyphae proliferated at a high rate during the infection process, which suggests the involvement of enzymatic activities (Veenhuis, et al., 1989). Some studies, however, have shown that these organelles are not involved in either the secretion of adhesives or the lysis of the cuticle (Jansson and Nordbring-Hertz, 1988). Protein changes of Caenorhabditis elegans and D. coniospora were observed during infection Coles, et al., 1989). H. rhossiliensis spores adhere to vermiform nematode cuticle before they penetrate the cuticle. A bulbous infection hypha formed from the spore and secondary hyphae developed from the hypha (Jaffee and Zehr, 1982). Some observations suggested that mechanical force is involved in the penetration of the nematode cuticle (Veenhuis, et al.,1985; Wharton and Mary,1990). The species of laptoglossa penetrates cuticle solely with mechanical force from the gone cell. Female and the cysts may be colonized by one of the following ways: (1)directly through the cuticle; (2) by penetration through the natural body opening; and (3) after colonization of the feeding cell within the root, which it may self cause the death of the female (Kerry,1988). Only a few fungi have been shown to penetrate the cuticular wall of nematode cysts. Probably, the first study of penetration of cyst wall by fungi was reported by Hanssler and Hermanns (1981) using transmission electron microscopy. In their study, V. lecanii penetrated cyst wall of H. schachtii only 48 hours after inoculation. It was concluded that lytic enzymes secreted by the fungus played a major role in its penetration of the cyst wall. Inside the cyst cavity, the fungus penetrated eggshells and colonized the juveniles (Hannssler, 1990). Kim et al. (1992) observed that a sterile fungus, designated as Arkanas Fungus 18 (ARF18), penetrated cyst wall and eggs of H. glycines Chen and Dickson (1996) examined 12 fungal species with the aid of light, and scanning and transmission election microscopy for their ability to penetrate the cyst wall of H. glycines. Nine of them penetrated the cyst wall from inside the cyst. At least three species, Exophiala pisciphila, Pyrenochaeta terrestris ,and Fusarium oxysporum , also penetrated the cyst wall from the outside. Generally, penetration could be completed by a single hypha. The penetration hyphal pegs within the cyst wall were generally less than 1µm (usually about 0.5 µm) in diameter, which is much less than the diameter of a regular hypha. Numerous organelles were observed in penetration hyphae of P. terrestris. When penetration of the cyst wall was initiated from outside to inside, hypha was observed attached to the cyst wall and appeared to dissolved a hole through the wall. The contents of the fungal cell with condensed organelles extended into the cyst wall, from which several branches of hyphae may have developed. At this stage, the wall of the penetrating hypha was not obvious. The penetrating hyphae of V. chlamydosporium in the cyst wall, however, appeared to have cell walls. The ability to penetrate the cyst wall from the outside may not be important for a fungous to colonize a cyst because fungi can readily enter cysts through natural body openings (Kerry,1988). In fact, fungi were usually found inside cyst cavities within 1 day after inoculation, before any direct penetration of the cyst wall was observed. The cuticular penetration may be important for the infection process to occur on young females. If a fungus invade can invade young females, the fungus may have more chance to destroy eggs within the cysts because eggs in the early developmental stage are more vulnerable to fungal infection (Chen and Chen, 2003; Irving and Kerry, 1986). Penetration of nematode egg by fungi is completed by either an appresorium or a simple hypha. Appressoria involved in penetration of nematode eggs have been observed in V. chlamidosporium (Lopez-Llorca and Claugher, 1990;Stirling and Mankaw,1979), Dactylella oviparasitica (Stirling and Mankau,1979), and P. lilacinus (Dunn, et al., 1982; Holand, et al., 1999). P. lilacinus was also able to colonize nematodes through simple hyphal penetratio (Dunn,et al., 1982). Zoospores of C. anguillulae encysted on nematode eggs by chance and germinated in such a way that the germ tube penetrated the lipid layer. Enzymatic and mechanical activities may be involved in the penetration of eggs, although the processes are poorly understood. Fungi without any chitinolytic activity probably penetrate only mechanically damaged eggs (Kunert and Lysek, 1987), while chitinolytic activity is probably important in dissolving the egg shell. Studies done by Segers et al. (1996, 1999) demonstrated that a subtilisin-like protease designated as VCPI was involved in infection of nematode eggs. The subtilisin was originally isolated from V. chlamidosporium, later also isolated from Metarhizium anisopliae, an insect pathogen, and V. lecanii, pathogen of nematodes and insect, but not from plant-pathogenic species of Verticilium, suggesting its role in the infection of invertebrates. Kunert and Lysek (1987) showed no evidence for the involvement of lipolytic enzymes in the penetration of the nematode egg shell by parasitic fungi. 2.6. 4. Pathogenicity of fungi to nematodes:- Once a fungus penetrates a nematode, the fungus usually proliferates within the nematode, eventually kills the nematode, and consumes the nematode body contents. The pathogenictiy, however, varies among nematophagous fungi. Among the predacious fungi, those that form unmodified adhesive hyphae or branches are considered primitive types, whereas fungi forming constricting rings are more involved and highly aggressive (Gray,1988). Ingestion of nematode by predacious fungi is often completed within 48 to 72 hours (Jansson and Nordbring-Hertz, 1988). Most endoparasites of vermiform nematodes are obligate parasites, and they are dependent on nematodes for nutrition. Cayrol and Frankowski (1986) demonstrated that a single H. rhossiliensis conidium attached to Ditylenchus dipsaci was sufficient for infection and causing death of the nematode. Some fungi isolated from cysts and eggs of Heteroderidae are saprophytic or weak pathogens. Many, however, are capable of parasitizing sedentary nematode females and eggs. Chen et al. (1996) studied 21 isolates of 18 fungal species for their pathogenicity to eggs of the H. glycines. The pathogenicity to the nematode eggs varied among the fungal species. Variation of host specificity and pathogenicity to nematodes has been observed among the isolates within a species of nematophagous fungi in many studies. Tedford et al. (1994) tested 25 isolates of H. rhossiliensis and divided the isolates into four groups according to there host preference. Liu and Chen (2001) tested 93 isolates of H. rhossiliensis for their pathogenicity to H. glycines J2 in the laboratory and in the greenhouse. Although most isolates parasitized high percentages of J2 on water agar and in soil in the laboratory, some of them were weak parasites of the nematode. Variations in pathogenicity were also observed among strains of egg parasitic fungi such as V. chlamydosporium (Kerry, 1987), P. lilacinus (Stirling and west, 1991), Cylindrocarpon desttructans (Rodriguez-Kabana and Morgan- Jones,1988), F. oxysporum, F. solani (Godoy, et al. 1982) C. anguillulae (Voss, et al. 1992) and Acremonium strictum (Nigh, et al. 1980).Variation in pathogenicity among isolates of a fungal species against nematodes appears to be the rule rather than the exception.
Only a few studies have been reported on biochemical processes of assimilation of nematode body contenrs by fungi. Hydrolytic enzymes are not likely to have a function in the digestion of nematode body contents. Oxidation of lipids, however, occurs later or during development of new vegetative mycelium (Jansson and Nordbring-Hertz, 1988).
2.7. Suppressive soils associated with fungal antagonists:- Nematode and their fungal antagonists in soil have co-evolved for a long period of time in natural history. The equilibrium between nematode density and some nematophagous fungi may exist in undistributed soil or in soil with continuing monoculture. A soil in which a nematode population on a susceptible cultivar maintains at a low level compared to an average infection level in a region can be called nematode suppressive soil. A nematode suppressive soil can be caused by biotic or abiotic factors. A low nematode population is often attributed to some factors such as resistance of host plants or soil environments. Only a few surveys have demonstrated the prevalence of natural biological control agents. The mechanisms resulting in nematode suppressive soils are poorly understood and extent and extent and frequency of natural occurrence are yet to be determined. It is difficult to quantify fungal parasitism of nematodes. Consequently, many reports on suppressive soils associated with fungi were observational rather than quantitative, and the conclusions made from some studies were superficial. A good example of nematode suppressive soils associated with fungal parasites is the decline of cereal cyst nematode in Europe. Reports from other nematodes and regions suggest suppressive soils are common in various agricultural systems throughout the world. 2.7.1. Decline of Heterodera avenae in Europe:- The decline of the cereal cyst nematode H. avenae has been well documented in Europe. This phenomenon was first demonstrated by Gair et al. (1969) in Britain. In a field infested by H. avenae, cereals were grown continuously for 13 years. The nematode population density peaked two years after the start of the experiment, then declined rapidly, and maintained low levels for the rest of years of the experiment. Similar population dynamics of H. avenae was observed in micro plot study (Kerry and Crump, 1998). This phenomenon was also observed from Germany (Ohnesorge, et al., 1974) and Denmark (Jakobsen, 1974). Application of formalin at 3000 liters/ha removed the suppressivnes, suggesting some biological factors were responsible for the decline ( Kerry and Crump, 1998). The decline of the cereal cyst nematode population has been attributed to fungal parasites of eggs and females. In Britain, major fungal species included N. gynophila, V. Chlamydosporium, C. destructans, and C. auxiliaries, but N. gynophila and V. Chlamydosporium were the most important, killing large number of females and eggs in many soils (Kerry, 1975; et al, 1982). In eastern Scotland, Verticilium sp., N. gynophila, Paecilomyces carneus, and C. destructans were the major species for controlling the population densities of H. avenae below economic threshold levels (Boag and Lopez-Llorca, 1989). 2.7.2. Suppression of Heterodera avenae in Europe:- The soybean cyst nematode occurs in most soybean producing regions. Decline of the soybean cyst nematode has been reported from the USA and China. Hartwig (1981) observed decline of H. glycines population density in breeding micro plots and field plots after 5 years of monoculture. He believed pathogens might suppress the nematode populations. But types of pathogens involved were not determined. Carris et al. (1989) surveyed population changes of the soybean nematode over 3 years in two Illinois fields. In one field the nematode maintained a low level without significant damage to the soybean even a susceptible soybean cultivar was grown continuously for 4 years before the study. In the other field, the nematode increased rapidly during the soybean-growing season. In the field with suppressed nematode population, the mycota was more diverse, and frequency of fungal colonization of cysts and frequency of pathogenic fungi were higher than that in the field with high nematode population densities. The data suggested that the fungi might be involved in nematode suppression. Soil suppressive to the soybean cyst nematode was also observed in a Florida field (Chen, et al., 1994, 1996). The soybean cyst nematode was introduced into the field in 1985 and the nematode population developed poorly (about one cyst/100cm3 soil in 1992) over 7 years of monoculture of soybean, suggesting the soil was suppressive to the soybean cyst nematode. Microwave heating treatment removed the suppression from the soil in a greenhouse assay. The nematode density was fourfold higher and the number of eggs produced by female was73% higher in microwave-heating-treated soil compared with untreated soil. The nematode population density was negatively correlated with both percentage of cysts colonized by fungi and fungal parasitism of eggs. A black, yeast-like fungus was frequently isolated from the soil and might be the major fungal parasite but not the only fungus responsible for the suppression of the nematode (Chen, et al.,1996). Decline of the soybean cyst nematode populations has also been observed in several locations in China (Liu and Wu, 1993). The nematode population densities increased in the first few years of monoculture and then declined thereafter to a level that had no significant damage to soybean. Three soil samples have been assayed in the green house to determine supressiveness to the nematode. The nematode female numbers on soybean roots increased 2.4 to 3.2 fold in soil treated with a fungicide, compared to untreated soil. Autoclaved soil mixed with untreated field soil at a ratio 9 : 1, indicating the suppressiveness can be transferred to the autoclaved soil. The results suggest that fungal antagonists of the nematode might be involved in the suppression of the nematode populations. While several fungi were common in cysts from two soils, a single predominant fungus, P. lilacinus, was observed in one soil. Other factors, however, may also be partially responsible for the suppression but have not been determined. 2.7.3. Suppression of Heterodera schachtii by fungi:- Suppression of H. schachtii by fungal parasites of eggs and juveniles has been observed in several locations in Europe and USA. In a field in the Netherlands, sugar beet was grown continuously from 1965 to 1982 (Heijbroek,1983). At first the population of H. schachtii was high but decline during the first few years of the trial. Although there was a later increase in number of nematodes, they did not reach the initial density. The most important fungal parasites in the field were V. chlamidosporium and C. destructans. The fungal parasitism of eggs reduced nematode population density. Suppression of H. schachtii population by H. rhossiliensis was reported in oil radish fields in Germany (Muller, 1982). The fungus parasitized as high as 90% of H. schachtii. Soil treated with fungicide reduced the fungal parasitism and increased the nematode population density. Although sugar beet cyst nematodes were suppressed by fungal pathogens, mainly C. destructans, V. chlamydosporium, N. gynophila, and C. auxilaris (Crump and Kerry, 1983), the soil containing these fungal pathogens did not cause H. schachtii population densities to fall bellow the damage threshold (Crump and Kerry, 1987) It appears that H. schachtii is not commonly under natural control by fungi as H. avenae is in Europe. Westphal and Becker (1999) demonstrated a soil suppressive to H. schachtii in a field in California. Treatment with fumigants or aerated steam reduced suppressiveness of the soil against H. schachtii. More fungal-infested females and cysts were found in the suppressive soil than a conducive soil (Westphal and Becker, 2001). The most common fungi isolated from the infested cysts were F. oxysporum, Fusarium sp. nov. , and D. oviparasitica. 2.7.4. Suppression of Meloidogyne spp. By D. oviparasitica:- Ferris et al. (1976) observed that root-knot nematode populations were low in some old peach orchards in California, despite the climate and host were suitable for the nematode developments. A subsequent survey led to the discovery of D. oviparasitica, a fungus parasitic to the eggs of root-knot nematode (Stirling and Mankau, 1978). The fungus parasitized 20% to 6o% of the eggs in the orchard throughout the year and reduced nematode numbers and root galling in a sterile soil in greenhouse (Stirling, et al. , 1979). A greenhouse study and the field observations suggested that D. oviparasitica was responsible for the suppression of root-knot nematode in the peach orchards.
2.7.5. Suppresion of Mesocriconema xenoplax by H. rhossiliensis:-
Mesocrionema xenoplax is one of several factors contributing to peach tree short life disease complex in southeastern USA. In some orchards, the nematode population declined unexpectedly even though the weather patterns and farm practices were suitable for the nematode. The observation of this phenomenon led to suggestion that some biological factors ma regulate the nematode population. A survey conducted in 22 peach orchards in south Carolina and one orchard in Georgia resulted in a discovery of frequent parasitism of nematode by H. rhossiliensis (Jaffee and Zehr, 1982) The nematodes parasitized by the fungus were often with brown heads and distorted hyphae-filled bodies. A greenhouse study showed that the fungus suppressed the nematode multiplication (Eayre, et al. , 1987). The fungal parasitism of the nematode may be partially responsible for the suppression of the nematode population in some orchards in southern USA ( Zehr, 1985). However, even in the presence of the fungus, nematode population densities were generally far above damage levels. Further studies on population biology of the nematode in California peach orchards suggested that the fungus might be a week regulator of M. xenoplax (Jaffee, et al. , 1989). 2.8. Potential Fungal Agents for control of nematodes:- Numerous fungi including various types of fungal antagonists of nematodes have been tested for their efficacy in control of plant parasitic nematodes. However, only a few fungi have been commercialized. And the product are not accepted for only used at a small scale. Before the 1970 s, most fungi tested for control of nematodes were predacious fungi and a few endoparasites of vermiform nematodes. It seems the predacious fungi are unlikely to be effective in control of nematodes as introduced agents into an agricultural cropping fields unless more efficient organisms are identified and better delivering systems developed. Recently, studies on nematode egg parasites and the fungi that produce toxins to nematode have been increased. Some of them have been shown to be promising in control of various nematodes. The following are a few fungi as examples that recently have been more or less extensively tested and have shown some potential in control of plant-parasitic nematodes. 2.8.1. Paecilomyces lilacinus Paecilomyces lilacinus is a typical soil-borne fungus that has been reported from numerous parts of the world, but it seems to be most frequent in warmer regions (Domsch, et al. , 1980). The fungus has been found from various types of habitats. Since Jatala et al. , (1979) discovered the infection of eggs of M. incognita and Globodera pallida and females of M. incognita. The fungus has been isolated from eggs, egg masses, females and cysts of many plant-parasitic nematodes through out the world. The fungus first colonizes the gelatinous matrix of Meloidogyne, Tylenchulus, and Nacobbus and cysts of Heterodera and Globodera; eventually a mycelial network develops and engulfs the nematode eggs. Penetration of nematode eggs is completed with an appressorium or simple hyphae (Holland, et al. ,1999; Jatala, 1986). Both mechanical and enzymatic activities may be involved in the penetration. Morgan-Jones et al. (1984) reported the fungal hyphae penetrated the egg shell of Meloidogyne arenaria through small spores dissolved in the vitelline layer. The fungus penetrate the eggs of Meloidogyne faster than the eggs of Globodera and Nacobbus because the eggshell of Meloidogyne is simpler than the eggshell of Globodera and Nacobbus (Rogers,1966). Following penetration, the fungus grows and proliferates in the eggs in early embryonic development. After depleting all nutrients in the egg, the mycelium may penetrate and rupture the cuticle of the infected egg from within and then emerge to infect other eggs in the vicinity. The fungus may also colonize the juveniles within the egg shell, and 3rd and the 4th stages of juveniles on water agar (Holland, et al. ,1999) Culture filtrates of P. lilacinus were toxic to nematodes (Cayrol, et al. ,1989; Chen, et al. , 2000; Khan and Goswami, 2000) Cuticles of nematodes were ruptured, and the nematodes were killed within a few hours after exposure to the culture filtrates. A peptidal antibiotic p- 168 has been isolated from culture of P. lilacinus and characterized (Isogai, et al. , 1980). This substance has anti-microbial activity against fungi, yeast, and therefore may enable the fungus to compete with soil microorganisms. P. lilacinus appears to be a good root colonizer (Cabanillas, et al. , 1988) and rhizosphere competitor. Its depth of distribution in sandy soils, however, appears to be limited to the upper 15 cm (Hewlett, et al. , 1988). The fungus can grow well over a wide range of temperature and pH and on various plant and animal substrates (Alam, 1990; Jatala, 1986). The fungus is also a parasite of insects. P. lilacinus has been tested widely for its potential as a biological control agent and shown to suppress nematode population densities and increase plant yields. Not all tests with P. lilacinus, however, resulted in successful control of nematodes (Hewlett, et al. , 1988). The variation of experimental results may be attributed to the variation of virulence among isolates (Stirling and West, 1991) and experimental conditions. A strain of P. lilacinus was shown to suppress M. incognita and M. arenaria populations but not Meloidogyne javanica (Wu, et al. , 1990). Combination of P. lilacinus with pasteuria penetrans increased efficacy in control of M. incogneta (Sosamma and Koshy, 1997; Dube and Smart, 1987). A formulation called (Biocont) containing P. lilacinus, was sold for control of root-knot and cyst nematodes in Philippines (Timm, 1987). A biocontrol agent named (Soybean Root Bio-Protectent) has been developed and used to control the soybean cyst nematode over 12,600 hectares in China (Liu, et al., 1996). The agent which contains P. lilacinus, organic materials, nematicidal plant broth, and mineral fertilizers, reduced H. glycines density and increased soybean yields (Wang, et al. , 1997). Although P. lilacinus has been isolated from human eyes and sinuses (Agrawal, et al. , 1979; Rokhill and Klein, 1980), there is no evidence that the isolates from nematodes are pathogenic to human. 2.8.2. Verticillium chlamydosporium:- Verticilium chlamydosporium has been known as a soil fungus since 1913 and is worldwide in distribution (Domsch, et al. , 1980). Since Willcox and Tribe (1974) discovered its parasitism of nematode eggs, the fungus has been found on various nematodes but mainly species of Heterodera and Meloidogyne. Gams (1988) reclassified the fungus into two species and two varieties of each species: V. chlamydosporium Goddard var. chlamydosporium, V. chlamydosporium var catenulatum. Cluster analysis of the enzyme activities indicated that some subspecific groupings of isolates may exist, but it did not support their division into two species ( Carder, et al. , 1993). The name V. chlamydosporium discussed herein represents the two species and four varieties. The ecology of V. chlamydosporium and biological control of cyst and root- knot nematodes were recently reviewed by Kerry and Jaffee (1997). V. chlamydosporium enters nematode cysts through natural openings or penetrate cyst wall directly (Kerry, 1988). The fungus forms branched mycelial network and penetrate eggs by simple branches of hyphae or by formation of appressoria (Lopez-Llorca and Claugher, 1990). Enzymetic activities are involved in penetration. Electron microscopy study showed that the fungus could disintegrate eggshell vitelline layer and partially dissolve chitin and lipid layers. A 32 kDa protease has been isolated from the infection of H. avenae eggs by V. suchlasporium and considered to be involved in the pathogenicity of the fungus to nematode eggs (Lopez-Llorca and Robertsin, 1992). An enzyme designated as VCPI from V. chlamydosporium can hydrolyze proteins from the outer layer of the eggshell of M. incogneta and expose its chitin layer (Segers, et al. , 1996). V. chlamydosporium may produce toxins that inhibit hatching or kill eggs of nematodes (Caroppo, et al. , 1990; Meyer, et al. , 1990). Juveniles within a cyst or egg mass may be colonized by V. chlamydosporium. Whether or not the juveniles were killed by toxin before the colonization is not clear. V. chlamidosporium also can parasitize females of cyst and root-knot nematodes (Kerry,et al. ,1982; Morgan- Jones, et al. ,) and other invertebrate animals such as snails. The ability of colonization of plant roots by V. chlamydosporium varies among fungal isolates and plant species (Bourne and Kerry, 1999; Bourne, et al. , 1994). Some studies showed that V. chlamydosporium are able to colonize plant roots (Kerry, 1984; Stiles and Glawe, 1989), while others indicated that V. chlamydosporium cannot invade the root cortex and is confined to the rhizoplane (De Leij and Kerry, 1991). The fungus appears to no pathogenic tendency to higher animals and humans. be non-pathogenic to plants (Kerry, 1984; Stiles and Glawe, 1989) and has V. chlamydosporium is one of major fungal parasites responsible for suppression of H. avenae in Europe (Kerry, 1975). The potential of the fungus in biological control of nematodes has been evaluated in many studies in greenhouse, microplots, and fields. The efficacy in control of nematodes is affected by several factors. Host plants have great influence on the growth of the fungus in the rhizosphere and the control efficacy (Borrebaeck, et al. ,1984). The fungus is more efficient in control of nematodes at lower nematode densities than at higher densities and in a poor host of nematode than in a susceptible host (Kerry and Jaffee, 1997). Root-knot nematodes in large galls may escape from the fungus attack, and control efficacy may be limited. Different isolates varied in their pathogenicity to nematode eggs (Irving and Kerry, 1986). A combination of the fungus with Pasteuria penetrans increased efficacy of reducing nematode population of M. incognita on tomato (De Leij, et al.,1992a).V. +chlamydosporium is a promising biocontrol agent, and efforts have been made to develop commercially acceptable formulation (stirling, et al., 1998). No commercial product from this fungus, however, has yet been on the market. 2.8.3. Verticillium lecanii :- Verticillium lecanii has been frequently isolated from soils in various geographic locations (Domsch, et al. , 1980). Besides its wide range of substrates of dead plants and animals, the fungus is a catholic hyperparasite and parasitizes arthropods, rust fungi, powdery mildews and many other fungi. It has been used commercially for control of greenhouse aphids. The fungus could penetrate cyst wall and colonize eggs of H. schachtii within 60 h (Hanssler, 1990; Hassler and Hermanns,1981). Lytic enzymes secreted by the fungus played a major role during its penetration of the cyst wall and eggshell. Gintis et al. , (1983) observed chitinase activity of the fungus on chitin agar and the ability to invade H. glycines eggs. Meyer et al. , (1990), however, reported that one strain of V. lecanii reduced viability of H. glycines eggs without colonization of the eggs, indicating that the fungus produced toxins that killed the nematode eggs. The fungus has been evaluated as a biological control agent against the soybean cyst nematode in the laboratory, greenhouse, and fields for several years. Benomyl-tolerant mutants were induced and a mutant was more efficient in reducing nematode populations of H. glycines and M. incognita in the greenhouse (Meyer,1994; Meyer and Huettel, 1991; Meyer and Meyer, 1995 and 1996). Application of alginate prills containing V. lecanii mutant strains at 5 g prills per pot (350g soil) significantly suppressed nematode population of H. glycines in non-sterilized soil, but no reduction of the nematode was observed with 0.5g prills per pot (Meyer and Meyer,1996). Microplot tests showed a significant control of H. glycines population with V. lecanii at 340 kg alginate prills/hectare; no control of nematode, however, was obtained in field plots (Meyer, et al. , 1997). More research is needed to determine whether or not the fungus has potential as a biological control agent. If the efficacy of control for nematodes is warranted, the fungus may be promising for control of nematodes at a large scale of field use. 2.8.4. Haustella rhossiliensis Haustella rhossiliensis was fist described in 1980 (Minter and Brady, 1980 ) based on a specimen collected from wales in 1953. sturhan and Schneider (1980) reported this fungus from the hop cyst nematode, Heterodera humuli, and named it as H. heteroderae (syn. H. rhossiliensis) The fungus has a wide range of hosts including plant-parasitic nematodes, free- living nematodes, entomopathogenic nematodes, and mites, although different isolates may have different host preferences, H. rhossilienis is a hyphomycete with simple erect phialides, which are swollen at base and tapering towards the apex. When a host nematode contacts the conidia on the phialides, the conidia may attach to the nematode cuticle, and infect the host nematode within a few days. Following penetration, the fungus forms an infection bulb in the nematode cavity, from which assimilative hyphae are developed. After converting nematode body contents to mycelial mass, the fungus may emerge from the nematode cadaver, produce spores, and infect other nematodes. An average of 112 conidia may be formed from mycelium developed from a single juvenile of H. schachtii at 20C (Jaffee, et al., 1990).KCl increased infection of nematodes by the fungus (Jaffee, and zehr, 1983) Conidia detached from the phialides may loss infectivity. Some conidia died shortly after sporulation and others may be viable and virulent for at least 200 days (Jaffee, et al. 1990). Variability of morphology, pathogenicity, and genetics was observed among isolates (Liu and Chen, 2001; Tedford, et al. 1994). Parasitism of nematodes by H. rhossiliensis is dependent on nematode density; the percentage of nematodes parasitized by the fungus is correlated positively with host nematode density (Jaffee, et al. 1992). The number of conidia attached to the cuticle of nematodes by H. rhossiliensis is correlated with the amount of conidia in the soil. Since the fungus is a poor soil competitor, local populations of the fungus may go extinct unless supplied with some minimum number of nematodes (the host threshold density ) (Jaffee and zehr, 1985). Natural epidemics of this fungus among populations of nematodes develop slowly and only after long periods of high host densities. Transmission of spores was greater in loamy sand than in coarse sand (Jaffee, et al. , 1990). In contrast to the theory that addition of organic matter may enhance activities of nematophagous fungi, addition of organic matter to soil decreased parasitism of M. xenoplax by H. rhossiliensis (jaffee, et al. , 1994). The potential of the fungus as a biological control agent has been controversial. Muller (1982) reported that the fungus might suppress cyst nematodes in some sugar beet fields in Germany. The fungus was considered partially responsible for suppression of M. xenoplax population in some orchards in southern USA (Zehr, 1985). High numbers and percentages of M. xenoplax parasitized by H. rhossiliensis were also found in some California peach orchards (Jaffee, et al. , 1989) In greenhouse studies, H. rhossiliensis suppressed G. pallida on potato (Velvis and Kamp,1996), H. schachtii on cabbage (Jaffee and Muldoon, 1989), Pratylenchus penetrans on potato (Timper and Brodie, 1994) and H. glycines on soybean (Liu and Chen, 2001). Results obtained by Tedford et al. (1993) indicated that long-term interactions between populations of H. rhossiliensis and cyst or root-knot nematodes did not result in biological control. In a field microplot test, H. rhossiliensis failed in suppression of H. schachtii (Jaffee et al. ,1996). The fungus, however, significantly reduced H. glycines in field plots (S. Chen, et al. , 1996; Lackey, et al. ,1993). More research, however, is needed to determine whether or not the fungus has potential as a commercial biological control agent. 2.8.5. Fusarium spp. Fusaium is a large genus including many species with a wide range of trophic adaptations. A number of Fusarium species have been isolated from females, cysts, egg masses and eggs of nematodes. F. oxysporum and F. solani are the most commonly encountered species. Strains of these two species are either phytopathogenic or non-phytopathogenic, but in general they are highly competitive in soil. Only a few species of Fusarium have been tested in laboratory and greenhouse for their potential biological control agents on nematodes. Nigh et al. ,(1980) demonstrated that a high percentage of H. schachtii eggs were parasitized by F. oxysporum in California sugar beet fields. Laboratory and greenhouse bioassays indicated that isolates from the fields are highly pathogenic to the nematode eggs. Chen et al. , (1996) reported that one isolate of F. oxysporum and one isolate of F. solani could colonize more than 30% and 20% of the eggs, respectively, in yellow females to light brown cysts of H. glycines on water agar. The same isolate of F. oxysporum colonized more than 70% of the eggs in newly formed females on roots in sterile soil in greenhouse pots. Species of Fusarium produce a large range of toxins, which are antagonistic to bacteria, fungi, and nematodes (Ciancio, et al. , 1988). Hallmann and Sikora (1994) reported that isolates of non-phytopathogenic endophytes, F. oxysporum, reduced root galls of tomato induced by root- knot nematodes by 52% to 75%. Culture filtrates of the non-phytopathogenic, endophytic isolates of F. oxysporum killed juveniles of M. incognita within 8 hours (Hallmann and Sikora, 1994). Nematicidal effect of culture filtrates was also observed from F. solani on M. incognita (Mani and Sethi, 1984). Toxins that are thermostable and independent of pH are responsible for the nematicidal effects. It seems that strains of non-phytopathogenic Fusarium species, which are either highly pathogenic to nematode eggs or produce metabolites toxic to nematodes, may exist in natural soil. Such strains plus their high competence in soil and rhizosphere may be effective biological control agents. 2.9. Ecology of nematophagous fungi:- An ecology study of nematophagous fungi conducted by Gray (1985) revealed that different types of nematophagous fungi have different edaphic preferences. The saprophytic nematophagous fungi that formed adhesive nets are found in soil with low organic matter and low moisture due to there saprophytic nature. When nutrients or moisture condition improved, the saprophytic nematophagous fungi are able to complete with other soil organisms by feeding on the expanding nematode population. In contrast, nematophagous fungi that form rings are more common in soil with high organic matter and moisture. Endoparasitic fungi that produce conidia are strongly influenced by organic matter. While most of the nematophagous fungi (except those that formed adhesive branches) are not affected by nematode densities, endoparasitic fungi that form ingestive spores are nematode – density dependent. In general, the conidia-forming endoparasites were isolated from samples with comparatively high soil moisture and low pH. Little is known about edaphic preference of the nematophagous fungi with unmodified adhesive hyphae, except that they are more frequently recovered from soils with higher pH. Based on their ecological preferences, nematode trapping-fungi are separated into two groups: saprophytic and parasitic nematode trapping- fungi (Cooke, 1963). Saprophytic nematode trapping- fungi form 3- dimensional network traps in response to the presence of nematodes. Under low nematode population densities, they remain saprophytic. Therefore, they are regarded as inefficient nematode- trappers. Parasitic nematode trapping-fungi have low saprophytic ability, but form traps spontaneously. This group consists of nematode-trapping fungi that form constricting rings, adhesive branches and are more effective nematode trappers than the saprophyticnematode-trapping fungi.(Jasson, 1982). 2.10. Taxonomy of nematophagous fungi
The bulk of the published work on the nematode destroying fungi consists of descriptions on the morphology with discussions on the classifications of the approximately one hundred and fifty described species. An excellent key to the identification of the nematode destroying fungi has been published by Cooke and Godfrey (1964). This key treats all of the fungi described to 1964,included all the common species and is accompanied by a comprehensive bibliography. Since the original descriptions of most of the described species there have been a number of taxonomic reappraisals by Soprunov (1958), Subramanian (1963), Cooke and Dickinson (1965), Rifai and Cooke (1966), Cooke (1969) and others. A number of new generic names have been introduced and many of the classical species have been moved around as new combinations to one or other of the existing or new genera. 2.11. Biology of nematode- destroying fungi:- 2.11.1. Spores:- In the nematode-destroying fungi spores may be sexual or asexual, large or small, wet or dry, pigmented or hyaline, septate or not, for dissemination or persistence, with or without flagella, thin walled or thick walled, and of variable and unusual shape. Some are violently shot off and others modified to inject a moving host with an infective particle. Size of conidia in fungi must always be a compromise. A conidium can not be so big that it is difficult to disperse or so small that it can not carry enough reserves to establish it self in the few substrate. It is very important for a conidium to be in the right place at the right time and the chances for success are increased by numbers. In general, the conidia of predatory fungi are quite large and those of endoparasites small. Large conidia require more energy input and fewer will be produced. Thus if a conidium is large this should be without purpose. Size of conidia in fungi must always be a compromise. A conidium can not be so big that it is difficult to disperse or so small that it cannot carry enough reserves to establish itself in the new substrate. It is important for a conidium to be in the right place at the right time and the chances for success are increased by numbers. We have noted previously that, in general, the conidia of predatory fungi are quite large and those of indoparasite are so small. Large conidia require more energy input and fewer will be produced. Thus if a conidium is large this should not be without purpose. Conidia of predators require substantial energy reserves since on germinations the conidium must produce a significant amount of hyphal development with one or several traps. The larger the spore the more extensive the hyphal system and the greater the number of traps produced. Thus a relatively large spore like that of A. anchonia can germinate to produce a hypha bearing mostly five but as many as seven constricting rings. A smaller spore such as that of D. brochobaga (Drechsler,1937) might only produce a single ring. The spore of predatory fungus must produce at least one organ of capture. In contrast to predators, the conidia of indoparasites are generally small and in some cases minute. As noted, the conidia lie round in the environment until they attach to or are ingested by a passing nematode. When they germinate, therefore, the substrate is immediately available. Only enough energy is required for the conidium to penetrate the body cavity and the spore can thus be very small. Predatory fungi have dry spores and in most species these are borne aloft on conidiophores presumably to aid in aerial dispersal.Conidiophores however, are not influenced by gravity but grow vertically with respect to the substrate which may in fact be horizontal if the conidiophores come out from the side. This perhaps will have some significance in their environment where they must often sporulate in cavities with little real chance for immediate aerial dispersal. In some predators a solitary conidium is produced on a slender stalk, in others a few conidia. The most prolific and perhaps handsomest of the predatory fungi is A. oligospora where sympodial growth at the apex produces clusters of conidia at intervals along the length of conidiophore. In endoparasites the conidia are usually produced mucus and probably dispersed by water or splash drops or transported to new sites on the bodies of unwitting mites or other soil animals. Here, the conidiophores are relatively short perhaps to increase the chances of encounter with the host or disseminating agents. Observations on A. oligospora and other species show that when conidia drop to the surface of an old plate, they quickly disintegrate. Even aerial spores seem to collapse rather readily. Cooke and Satchuthananthavale (1968) had shown that germination hyphae from predatory spores frequently lyse and die in natural soils. Conidia may be a ready food source for soil animals. Thus, persistence of predators and parasites of nematodes under conditions of adversity such as cold or heat or drought or lack of a suitable host must depend on spore types other than conidia or encysted zoospores. 2.12. Biological control:- Nematode-destroying fungi are extremely common in natural soils, agricultural soils and all kinds of rotting organic debris (Duddington, 1951,1954) Most predators and certain of the endoparasites are not specific, and any particular fungus may attack a wide range of nematode species. It is natural therefore, that nematode- destroying fungi should be considered as agents for biological control. The history of attempts to use predatory fungi to control plant parasitic nematodes has been admirably summarized by Duddington (1962).The classical work in this area was carried out in Hawaii by Linford (1937) and Linford et al. (1938). These workers added chopped, green, pineapple tops to eelworm infested soil in pots and estimations were made of the nematode populations and the activity of the predatory fungi. It was established by Dobbs and Hinson (1953) that the spores of most fungi when placed in contact with natural soils show an inability to germinate under conditions which might be considered favorable for germination. This soil mycostasis is characterized by inhibition of spore germination. Although germinated conidia are less susceptible to soil mycostasis, nevertheless , considerable lysis and/or death of germinated spores may also occur. Mankau (1962) studied soil fungistats in Southern California soil with respect to the nematophagous fungi. Results showed that there was a water diffusible substance in all of the soils tested which inhibited germination of the Deuteromycetes, A. dactyloides, A. arthrobotryoides and D. ellipsospora. From his observations on germ tube and hyphal development on agar discs in contact with soil, Mankau concluded that nematophagous fungi are poor competitors in soil and suggested there ability to grow through soil in a saprophytic phase may be limited. He found that, in highly antagonistic soils conidia generally formed traps immediately on germination and then were entirely dependent on capturing nematodes for survival. Thus A. dactyloides produced a single constricting ring directly from the spore and A. arthrobotryoides produced a short adhesive germ tube. This ability of conidia of predaceous fungi to germinate directly noted by Mankau was also reported by Perrin (1972), for A. conoides where spores in droplets of condensate (on Petri dish lids) containing nemas germinated directly to produce an adhesive bud. This same phenomenon had been noted for several Arthobotrys species including A. dactyloides and A. conoids and D. brochopaga. Conidia of Dactylaria spores in contact with active nematode cultures will often germinate immediately to produce an adhesive knob from the end of the spore or sometimes from each end of the conidium. Mankau noted that all of the soils he tested contained nematodes. It is possible that mycostasis inhibits normal spore germination while the nematodes present produce morphogenic factors which stimulate immediate trap formation. Sensitivity to mycostasis by nematode-trapping fungi was studied by Cooke and Satchuthananthavale (1968) using fifteen species of predatory fungi. They found that with the exception of A. musiformis all species show sensitivity to mycostasis and the degree of inhibition varied from species to species. They also showed that successful germination of conidia in soil may not lead to successful establishment of the fungus as other antagonistic factors in soil may not lead to successful establishment of the fungus as other antagonistic factors in soil may have inhibitory effects on germinated spores. They concluded that, although agricultural soil frequently contains an indigenous population of nematode-trapping fungi, it would be difficult to increase the population significantly by adding spores of predators. They suggested that attempts to establish these fungi in agricultural soil for the purposes of biological control of eelworm pests are unlikely to meet with success. 2.13. Chemical attractants:- It is an attractive hypothesis to think that fungi produce chemical substances that lure nematodes to the site of trap formation in predatory fungi or to the site of spores or spore production in endoparasitic species. It is known that nematodes consume fungal spores as a normal part of their diet although these are not always digested (Jensen, 1967; Wasilewska and Webster, 1975). In experiment with Harposporium helicoids, Barron (1970) found that Rhabditis nematode readily consumed spores of the parasite. A single nematode sometimes swallowed over a hundred spores in the space in few hours. There is apparently a chemical attractant at work when Rhopalomyces elegans parasitizes nematode eggs. The conidia of Rhopalomyces on germination produce an extensive net of very fine sparingly branched hyphae. If an egg laid in the vicinity of hyphae, one or several hyphal branches quickly developed and grow more or less directly towards the egg and produce an appressorium on contact with its shell. These short branches are only produce in response to the presence of the nematode egg. It is presumed that branch growth is in response to exudates from the egg. It is possible that more than one chemical is involved; one to stimulate branch formation and another to control direction of growth. The stimulus causing the branch tip to swell up on contact with the shell is not known but may well be a contact response. In C. anguillulae, the zoospores tend to encyst at or near the body orifices. Sayre and Keeley (1969) noted that adult nematodes are more readily parasitized than larval stages and suggested that the larger and better developed body openings of adults allow the zoospores to accumulate and penetrate. It is likely that large orifices will allow substantial leakage of chemical substances to act as directional stimuli. Monoson and Ranieri (1972) believed that movement of Aphelenchus avenae nematodes around the traps of A. musiformis was not random and suggested that the animals were attracted to the trapping structures of the fungus. They attempted to prove that extracts of the fungus contained a nematode-attracting substance (NAS). Their results showed that extracts of A. musiforms, in which traps had been induced, produced a substance that aided in the attraction of nematodes. It was unfortunate that they used a fungal-feeding nematode and there results could be interpreted to show that a fungal-feeding nematode is attracted to fungal extracts. It was established by Townshend (1964) that A. avenae was attracted to 57 of 59 fungi tested. Monoson et al. (1973) compared extracts from A. musiformis hyphae with extracts from similar hyphae in which nets had been induced. Their tabular data showed that A. avenae nematodes were attracted to extracts obtained from hyphae with nets.
Balan and Gerber (1972) studied nematode attraction using the constricting ring predator A. dactyloides and the free-living nematode Panagrellus redivivus. They demonstrated a definite attraction of P. redivivus to filtrates of A. dactyloids cultures and found over six times as many nematodes were recovered using filtrates obtained from 4-day- old cultures as in the control. When homogenized broth and mycelium were tested, the attraction (over2×increase) was not as pronounced. They concluded that ammonia or some other substance counteracting attraction was released from the mycelium during blending. In attempting to characterize their attractant they found that the attraction could in part be explained by CO2.
It is well established that CO2 play as an important role in attracting nematodes (Nicholas,1975). In the natural system hyphae and nets of Arthrobotrys are rather sparing and it seems unlikely that such sites would be responsible for high levels of CO2 output as compared with the metabolic activities of dense population of other microbial life forms particularly bacteria. Response of a nematode to a CO2 gradient would likely lead to a food source rather than a predator. Klink et al.(1970) studied host finding mechanisms in the plant parasitic nematode Neotylenchus linfordi and found that nematodes congregated around colonies or filtrates from mycelium of several species of fungi tested. They found that filtrates from Gliocladium roseum were the most active and identified the attractants as small thermostable molecules. It is possible that similar mechanisms of attraction might be operative for free- living nematodes and the nematode-destroying fungi.
Balan and Gerber (1972) suggested from their experiments that ammonia produced by A. dactyloids was responsible for the death of captured nematodes. Using bromthymol blue as an indicator, they demonstrated that newly formed traps and parts of the hyphae stained blue, indicating possible sites of ammonia production. Their chemical tests for ammonia were positive, both in dishes where trap formation was not induced. Katznelson and Henderson (1963) showed that Rhabditis nematodes were attracted to certain soil and fungi and Actinomycetes and suspected ammonia production by the organisms as being responsible. The test showed that ammonia chloride (50ug/ml) produce an effect equal to that of the culture filtrate. On the face of it, the results of Katznelson and Henderson (1965) seemed to conflict with those of Balan and Gerber (1972). The levels of ammonia, however, were much different and the possibility exists that low levels of ammonia might attract nematodes and, following capture, high levels of ammonia might deactivate the prey. 2.14. Specificity:- Drechsler (1941) in his studies on the specificity of a particular parasites to a particular host and in his wide ranging and critical studies on nematode- destroying fungi, frequently made observations regarding specificity of fungi to their nematode hosts. He noted that in some cases the fungi apparently confined there attack to a single species of nematode whereas in other cases a range of nematodes was attacked. Also, where more than one species was attacked the fungus sometimes showed greater virulence against one species than another. Drechsler noted that Haptoglossa heterospora and Meria coniospora destroyed many nematode species. Meristacrum asterospermum attacked species of Rhabditis, Aphelencoides and Plectus parcus. This was also true for Harposporium helicoids and H. oxycoracum. On the other hand, H. diceraeum apparently attacks P. parcus most often and H. baculiforme apparently confined its attack to a single species of Plectus. Birchfield (1960) showed that C. vermicola had a wide host range and attacked eleven species of plant parasitic nematodes although ring nematodes of the family Circnematodae were more resistant than the others to infection. Similarly C. anguillulae attacks many hosts and in studies on its pathogenicity against selected nematodes, Esser and Ridings (1973) listed host species thirteen genera that were susceptible to attack and species from eleven genera that were not attacked. Sayre and Keeley (1969), working with C. anguillulae, noted that adult nematodes are more susceptible to attack than younger nematodes. They attribute this to the larger body openings of the adults which probably allow the fungus to accumulate and penetrate. In comparative studies, they found that Panagrellus redivivus was more susceptible than Ditylenchus dibsaci but pointed out that such differences in susceptibility may be due to the fact that P. redivivus was under considerably more physiological stress under their experimental conditions. Regarding predaceous species, certain types of trapping devices would be expected to be non-specific as in the case with rings and constricting rings. The latter will responds to a mechanical stimulus and will close on a glass rod if properly triggered. Such devices to be non-specific and trap any nematode that touches the inside of a ring would be expected. On occasions rotifers can be found caught and killed by constricting rings. The hyphal development in such rotifers may not be particularly suitable sources of nutrient. It may be that some nematodes, although captured, may not be invaded extensively by the fungus. It is possible that some nematodes may have developed antibiotic capabilities to counter attack fungal invasion. As pointed out by Drechsler (1941), in general the adhesive organs of capture show little discrimination in their choice of prey except such as may result from the physical limitations of their predatory apparatus. Nevertheless, observations show that some nematodes seem unaffected by the nets of certain fungi.
CHAPTER THREE MATERIALS AND METHODS 3. 1. Sampled sites:- Sampling sites were chosen to represent particular areas under different agricultural practices in the Agricultural Research Farm (ARC), Bashkar area (Elmuslamia group), Wadi shaaeer, Om Sunt, Hantoup and ARC Cattles Farm. Sites chosen were those have been infested with soil borne diseases and nematodes. Sampled sites from each locality were as shown below :- 1- An area cropped to tomato ( Lycopersicon esculentum ) 2- " " " " carrot (Daucus carota) 3- " " " " lemon (Citrus aurantifolia) 4- " " " " banana (Musa SP.) 3.1.1. Sampling method :- Soil samples, ¼kg each were collected from several sites in the particular locality by scrapping the surface and subsurface area to a depth of about 10 to15 cm, as most of soil borne fungi and nematodes are usually found at this layer. Soil samples from each site were thoroughly mixed with the aid of a sterile trowel and shovel and reduced by quartering to about 25g. Samples were collected in polyethylene bags, labeled, transported to the laboratory and kept in a refrigerator for further study. 3.2. Methods of isolation of soil fungi:- Corn meal agar (CMA) was prepared ,sterilized, cooled and poured into 9 cm diameter Petri dishes. Many plates were prepared for each sample. Soil collected from different plants was sprinkled lightly on the media. The Petri dishes were sealed, labeled and incubated at 25ºC and then investigated after one week and the investigation went on daily for several weeks for the appearance of dead nematodes. 3.2.1. Media used:- 1-Corn Meal Agar Corn Meal 200g Agar 20 g Distilled water 1000 ml 2- Potato Dextrose Agar ( PDA ) Potato 200g Dextrose 20g Agar 20g Distilled water 1000 ml 3.2.2.Examination of plates and recovery of fungal isolates and nematodes:-
The prevalence of fungal colonies was followed from the seventh day. Periodic examination and sub culturing of fungal isolates, further identification were made.
3.2.3. lsolation of fungi from around the dead nematode:--
Hyphal tips were carefully picked by sterile needle from the growth of the colony which had been grown on the Corn Meal Agar media (CMA) and transferred to Petri dishes containing Potato Dextrose Agar (PDA).
The isolated fungi were transferred to small vials containing PDA with sloping surface and allowed to grow for one week. 3. 3. Identification of the isolates:- 3. 3.1. Identification of Trichoderma isolates:- The morphology of the colonies was recorded and then microscopic examination was made by mounting a small pieces of the colony in lacto phenol- cotton blue. Measurements of hyphae, conidiophores and conidia were taken and compared with the available monographes (Alexopoulous 1962), (Rifai 1969), (Ainsoworth et al. 1973), (Lilian 1974 ), (Webster 1978 ), (pitt 1979 ) and (Onions et al. 1981 ). 3. 3.2. ldentification of Arthrobotrys oligosora isolates:- Evaluation of the shape and colour of the aerial mycelium and conidia were microscopically performed. Small pieces of the fungal growth were mounted on either a drop of sterile distilled water or lactophenol cotton blue in order to simplify recognition of morphological structure. The mounts were covered with cover slips and examined under the light microscope. 3. 4. Preparation of the fungal inocula :- 3. 4.1. Preparation of the fungus Trichoderma harzianum and counting the spores:- The fungus T. harzianum which was isolated from around the dead nematode was transferred to Petri dishes containing potato dextrose agar culture medium. The plates were then incubated at 25±2ºC for 3-5 days. Fungal colonies grown on the plates were separately sub cultured into new PDA plates. The cultures of fungi which appeared on the new plates were purified by the single spore culture and hyphal tip method. After the plates covered the fungus, the spores the spores were harvested with sterillised with bruch and watered in large beaker. The collected spores were thieved to separate the spores from other debris. The clean spores were stirred to giv homogenized suspention and then a test tube was filled with the homogenized spores. A total spore count was made in an improved Neubauer haemocytometer. The homogenized spores in the test tube were diluted as required. The fungal stock culture kept at the plant pathology lab for further studies. 3. 4.2. Preparation of the fungus Arthrobotrys oligospora and counting the spores:- Also here, the fungus A. oligospora which was isolated from around the dead nematode was transferred to Petri dishes containing potato dextrose agar culture medium and incubated at 25±2ºC for 3-5 days. After the appearance of the culture of the fungus on the plate, the culture was purified by a single spore culture method.The same method of counting the spores had been done as mentioned in 3.4.1. The two fungi, A. oligospora and T. harzianum that had been identified were kept for further use against the different nematodes which had been trapped by them in different way of trapping. The fungus T. harzianum was used against Meloidogyne sp. and the fungus A. oligospora was used against Radophillus similis and Xiphenema sp. 3. 5. Preparation of nematodes:- 3. 5.1. Collection of infested soil samples:- Infested soil samples were collected from soil around lemon, tomato and banana plantation. The depth of sampling was 10-15 cm since most of the nematodes are found in this layer. The soil samples were put in polyethylene bags, and then transferred to the laboratory for nematode extraction. 3. 5.2. washing and extraction of nematodes:- The Baermann - funnel technique was used for extraction of the nematodes, Guima and Cooke (1972). Before washing, each soil sample was put on thick polyethylene sheet and homogenized several times. 100 g of soil was moistened by adding 200ml of water in1000 ml beaker and 600 ml of water later added. The soil sample was thoroughly shaken and left for half an hour. Using tap water, the moistened soil was passed through a sieve 125 mm to separate large soil granules and other soil debris. Soil surface was filtered several times to ensure maximum collection of nematodes. The supernatant was decanted in 38 mm sieve and carefully washed with tap water to get rid of mud to ensure clean nematodes. The residue containing living nematodes was collected in a 100 ml beaker and poured into a 5 inch diameter funnel containing water, the funnel was covered with a muslin and/or double play Kleenex (to trap the left debris). Rubber tubing 15 cm was fitted into the stem of the funnel with a clip at its end. Then living nematodes swim and pass freely downwards. After 24 hours the nematodes accumulated in the stem of funnels. The nematodes were collected in test tubes and labeled. 3. 5.3. Estimation of living nematodes:- Nematode collected from different soil type was counted. Living nematodes in the test tube which extracted from 100g of soil as mentioned before were transferred in the counting dish using stereomicroscope. A number of 50 different nematodes under study were prepared separately for further study. 3.6.Glasshouse experiments:- 3.6.1. Experiment (1) :- Plastic pots (12-inch internal diameter) were filled with sterilized soil and inoculated with suspension of different concentrations of the already prepared Trichoderma, the concentrations used were 10-1, 10-2 and 10-3 The standard solution was 106 spores/ml. Four pots were used for each treatment. The inoculum was thoroughly mixed with the soil. Five seedlings of tomato (Lycopersicon esculantum) variety Strain-B were directly transplanted in each pot. A number of 50 Meloidogyne spp nematodes were added to each pot. Some pots kept free of inoculum as control. The pots kept under glass house conditions and watered regularly and they arranged in an order of completely randomized block design with three replications. Number of leaves and plant height were measured every month. 3.6.2. Exp. (2):- The same method of experiment (1) which mentioned above was used, but in this one the soil was inoculated with the suspension of different concentrations of the already prepared A. oligospora, the concentrations used were 10-1, 10-2 and 10-3. Seedling of lemon at the same age were sown and 100 number of Xephenema sp. were added to each pot. Also number of leaves and plant height were measured every month.
3.6.3. Exp. ( 3 ) :- Banana seedling were sown directly in clay pots containing a sterilized soil (mixture of Gezira clay and sand with a ratio of 1:2 respectively). The soil inoculated with suspension of different concentrations of the already prepared A.oligospora The concentrations used were 10-1, 10-2 and 10-3 . The standard solution was 106 spores/ml. The inoculum was thoroughly mixed with the soil. Radophilus sp. nematode (100n) were added to each pot. Some pots kept free of inoculums as control. The pots kept under glass house conditions and watered regularly and they were arranged in an order of completely randomized design with three replications. Number of leaves and plant height of each plant were measured every month. After 3 months, 5 months and 6 months, plants of tomato, banana and lemon respectively were carefully removed and washed in running water then placed in plastic bags with labels and then taken to the laboratory, they were dried and the following parameters were measured and the averages were calculated:- Fresh root weight Fresh shoot weight Dry root weight Dry shoot weight In all the experiments, number of nematodes was calculated in each pot after the removal of the plants using Baermann- funnel technique for the extraction as mentioned before.
3.7. Metabolites of the fungus Tricoderma harzianum 3.7.1. Medium preparation and inoculum Media were prepared in one liter Erlenmeyer flasks, according to the method of Elad et al. (1982). The medium contained 0.2g magnesium sulphate (MgSO4.7H2o); 0.9g dipotassium hydrogen phosphate (K2HPO4);0.2 potassium chloride (KCL);1.0g ammonium nitrate (NH4NO3); 0.002g ferrous
(Fe2 +); 0.002g manganese (Mn2+); 0.002g zinc(Zn2+), per liter of distilled water. The medium was transferred into Erlenmeyer flask. The flasks were plugged with cotton and sterilized at 121 ºC for 20min. After sterilization the flask was incubated with a five mm diameter agar plug cut with sterilized cork-borer from the edge of a 7 day- old culture of the T. harzianum. The flasks were incubated at 25º C for 7 days. The method of Brewer et al. ( 1982) and Elad et. al.(1982) was adopted for extraction of the metabolites. The culture of the Trichoderma was collected in beaker (1 liter) and then blended with a kitchen blender for 20 minutes. The fungal growth was separated from the filtrates by centrifugation at 3000 rpm for 20 minutes and filtered by passing through micro bore filter (0.2u). The supernatants of the T. harzianum was collected into volumetric flask (250ml), cooled at 4.0º C in a refrigerator for 24 h. Methyl acetate (100ml) was added to separate the metabolites. The water was separated from the mixture in a separatoring funnel. 3.7.2. Purification of the metabolites The separated methyl acetate was evaporated at 50º C using a rotary evaporator. The residue was dissolved in a mixture of methyle alcohol ( 100.0ml), petroleum ether (100.0ml). The phases were separated using a separatoring funnel. The metabolic phase was extracted three times with the Petroleum ether and the combined petroleum ether extract were shaken with 95% (v/v) aqueous methyl alcohol (100ml). The combined solutions were concentrated at 20 ºC by a rotary evaporator until most of the methyl alcohol and petroleum ether were removed. The remaining concentrated solution was kept as stock solution in a 10.0 ml volumetric flask for further study. 3.7.3. Effects of the metabolites on the nematode Sample of methyl alcohol petroleum ether containing the Trichoderma metabolites prepared as mentioned above was distilled in a rotary evaporator at 50º C until all methyl Alcohol was removed. The residues were then dissolved in 10ml distilled water and different dilution of the metabolites (10-1, 10-2, and 10-3) were then prepared. Petri dishes were sterilized and a number of 50 Xiphenema sp. was put in each dish. An amount of 2 ml of each dilution of the metabolites was transferred into each Petri – dish separately. Distilled water was used in certain Petri-dishes instead of the metabolite as control. The experimental design selected was arranged in an order of completely randomized block design with three replications. Number of dead nematodes was calculated after 24, 48 and 72 hours.
CHAPTER FOUR
RESULTS
4.1. Preliminary examination and observations of nematophagous fungi before identification:-
During the periodic examination of the Petri dishes which had been inoculated with the different samples of soil that collected from different plants, lemon, tomato and banana colonies of unknown fungi had been seen and also unknown free living nematodes had been seen attacked with those fungi in different way of trapping using a digital microscope (Digital Blue QX5 computer microscope). Fig. (1and2) showed that how the fungus trapped the nematode by net. Fig. (3and4) showed how the nematode had been trapped by adhesive knob and also there is a knob seen on the left side of the picture. Fig. (5) showed the spores of the fungus inside the attacked nematode. In Fig. (7) The fungus inside the host body and all the body contents had been consumed as can be noticed. Fig. (8) and Fig. (9) showed that nematodes which had been dead without any way of trapping, thus they had been dead with the toxin of the fungus Trichoderma. Fig. (10) showed that how the spores breaking out through host cuticle. 4.1.1. Isolation of fungi from around the nematodes:- Two fungi which had been observed on different Petri dishes had been identified after they were isolated from around the different dead nematodes. One of them identified as Trichoderma harzianum and the second one identified as Arthrobotrys oligospora.
Fig. (1) :- Nematode captured by adhesive net and had been held at least at two points
Fig. 2:- Nematode trapped by net and had been attached at several points.
Fig. 3:- Nematode captured by adhesive knob, the knob is seen left of picture.
Fig. 4:- The nematode struggled after capturing and dead.
Fig. 5:- The spores of A. oligospora inside the dead body of the nematode
Fig. 6:- The mycelium and spores of the fungus A. oligospora.
Fig. 7:- The mycelium inside the host body and all the body contents had been consumed.
Fig. 8:- Nematode on agar plate dead with the toxin of Trichoderma.
Fig. 9:- Dead nematode with the fungus Trichoderma.
Fig. 10:- Spores of a fungus penetrating out through nematode cuticle.
Fig. 11:- Inside the host body had been consumed by the fungus and the spores arise out of the host corpus. 4.1.2. Identification of Trichoderma isolates:- The shape and colour of the aerial mycelium was evaluated macroscopically. The colour of the culture is green. The conidiophores hyaline, much branched, not verticillate ; conidia hyaline, 1-celled, ovoid, borne in small terminal clusters; usually easily recognized by its rapid growth and green patches or cushions of conidia. 4.1.3. Identification of Arthrobotrys isolates:- The shape and the colour of the conidiophores and the conidia was evaluated macroscopically. The conidiophores are long, slender, simple, septate, hyaline, slightly enlarged at the apex and spore-bearing regions. Also conidia hyline, unequally 2-celled and ovate-oblong. 4.2. Preparation of nematode under test:- The nematodes which prepared to use them for this test were Meloidogyne Javanica, this one attacks tomato crop. The other one is Xiphenema sp. Which attack Lemon and also Radophillus similis which attack banana. 4.3. Glass house experiment:- 4.3.1. Experiment (1) 4.3.1.1 Effect of different concentrations of the fungus T. harzianum inoculums on number of tomato leaves:- In the first season, (Table 1) showed that, the different concentrations of the fungus inoculums significantly increased the number of leaves from the first month of the observation up to the six month compared to the untreated control. In the second season, the different concentrations of the fungus inoculums significantly increased the number of leaves through out the season from the first month up to the six month as shown in (Table 2).
Table 1:- Effect of different concentrations of the fungus T. harzianum inoculua on number of tomato leaves season (1) :-
Treatments after 1 after 2 after 3 after 4 after 5 after 6 spores/ml month month month month month month
105 8.33a 12.67 a 13.00 a 11.50 a 17.25 a 18.25 a
104 5.33b 6.00 b 8.00 b 11.25 a 16.75 a 17.75 a
103 4.33bc 5.00 b 6.00 c 11.00 a 16.25 a 17.50 a
Control 3.67 c 3.93 b 4.00 c 5.07 b 8.66 b 6.92 b
CV% 10.66 25.97 10.83 9.01 10.34 7.76
SE± 0.33 1.05 0.500 0.59 1.00 0.80
Means in the same column followed by the same letter are not significantly different at P≤ 0.05. Table 2:- Effect of different concentrations of the fungus T. harzianum inoculua on number of tomato leaves season (2):- Treatments after 1 after 2 after 3 after 4 after 5 after 6 month month month month month month spores/ml
105 5.25a 7.75 a 8.75 a 10.00 a 12.00 a 10.00 a
104 5.00 a 7.00 a 8.25 a 9.75 a 11.50 a 10.00a
103 4.75 a 6.50 a 8.00 a 9.50 a 11.00 a 9.50 a
Control 3.48 b 4.34 b 5.59 b 6.07 b 4.71 b 5.00 b
CV% 19.15 15. 07 13.42 12.44 8.20 10.17
SE± 0.55 0.62 0.65 0.70 0.54 0.58
Means in the same column followed by the same letter are not significantly different at
0.001 ml Control 0.1 ml 0.01 ml
Fig. 12 The effect of different concentrations of the fungus T. harzianum inocula on the vegetative growth of the tomato plants 4.3.1.2. Effect of different concentrations of the fungus T. harzianum inoculums on stem length of tomato:- All treatments significantly increased tomato stem length compared to the untreated control up to the six observation in the first season, (Table 3). In the second season, The high concentration of the fungus inocula significantly increased the stem length compared with the medium and lower dosage rates of the inoculua and the untreated control, (Table 4)
Table 3:- Effect of different concentrations of the fungus T. harzianum inoculua on stem length of tomato season (1) :-
Treatments after 1 after 2 after 3 after 4 after 5 after 6 month month month month month month spores/ml
105 17.00 a 21.00 a 25.25 a 28.25 a 31.75 a 33.25 a
104 16.75 a 19.25 a 22.25 ab 24.25 ab 30.75 a 32.00 a
103 14.25 a 17.25 a 17.50 b 21.50 b 28.25 a 30.50 a
Control 1.03 b 12.21 b 15.61 c 15.07 c 17.24 b 1.38 b
CV% 12.88 12.63 14.41 12.22 11.40 8.63
SE± 1.19 1.40 1.81 1.74 1.99 1.38
Means in the same column followed by the same letter are not significantly different at P≤ 0.05.
Table 4:- Effect of different concentrations of the fungus T. harzianum inocula on stem length of tomato season (2) :- Treatments after 1 after 2 after 3 after 4 after 5 after 6 month month month month month month spores/ml
105 17.67 a 21.00 a 26.33 a 28.67a 29.67 a 32.00a
104 13.00 b 15.33 b 22.33 a 23.00 b 25.00 b 26.67 b
103 9.00 c 10.67 c 13.67 b 15.00 c 16.00 c 18.00 c
Control 6.67 c 8.33 c 11.00 b 11.33 c 11.67 d 13.67 d
CV% 11.78 10.98 9.95 10.22 9.95 7.71
SE± 0.79 0.88 1.18 1.15 1.18 1.00
Means in the same column followed by the same letter are not significantly different at P≤ 0.05.
4.3.1.3. Effect of different concentrations of the fungus T. harzianum inoculums on tomato root length, fresh root weight and dry root weight:
In the first season, the high concentration of the fungus inoculums (0.1) gave significantly (p≤ 0.05) the highest root length over the other two rates (0.01, 0.001) and the control. Also it gave significant difference on the highest mean root fresh and root dry weight over the control and the other two rates, (Table 5 ). In the second season, also the high concentration of the fungus inoculums gave significantly (p ≤0,05) the highest root length over the third concentration (0.001) and the control. There is a difference in root length between the first concentration and the second concentration but it is not significant, (Table 6). Table 5:- Effect of different concentrations of the fungus T. harzianum inocula on tomato root length, fresh and dry root weight, season (1):-
Treatments Root length Root fresh Root dry weight weight spores/ml
105 24.00 a 6.37 a 1.43 a
104 19.00 ab 4.10 b 1.16ab
103 14.67b 2.69 c 0.83ab
control 6.33 C 1.05 c 0.27 b
CV% 22.70 31.67 32.10
SE± 2.12 0.65 0.17
Means in the same column followed by the same letter are not significantly different at P≤ 0.05.
Table 6:- Effect of different concentrations of the fungus T. harzianum inocula on tomato root length, fresh and dry root weight, season (2)
Treatments Root length Root fresh Root dry weight weight spores/ml
105 14.00 a 2.88 a 0.62 a
104 10.67 ab 1.51b 0.42ab
103 7.67 bc 1.00 b 0.28 bc
Control 5.33 c 0.86 b 0.19 c
CV% 18.31 22.31 27.40
SE± 0.10 0.20 0.06 Means in the same column followed by the same letter are not significantly different at P≤ 0
Fig. 13:- Effect of T. harzianum inocula on the incidence of root knot Control nematode on tomato. Treated
4.3.1.4. Effect of the fungus inocula on number of nematode in the soil after removal of plants under test:-
In both seasons number of nematodes which had been added to the soil with the different concentration of the fungus T. harzianum inoculums before transplanting tomato crops significantly reduced compared with the untreated control after the removal of the plant at the end of the experiment (Table,7 and 8).
Table 7:- Effect of the fungus inocula on number of nematode in the soil after removal of plant under test, season (1):-
Treatment Number of Number of spores/ml females males
105 11.67 b 78.67 b
104 31.00 b 109.30 b
103 43.67 b 173.70 b
control 147.00 a 516.70 a
CV% 69.65 71.28
SE± 23.46 25.5
Means in the same column followed by the same letter are not significantly different at P≤ 0.05.
Table 8:- Effect of the fungus inoculums on number of nematode in the soil after removal of plant under test, season (2):-
Treatments Number of Number of males females spores/ml
105 11.00 b 88.00 b
104 29.67 b 204.70 b
103 27.33 b 1.5.70 b
control 102.3 a 281.70 a
CV% 59.75 24.83
SE± 14.69 20.79
Means in the same column followed by the same letter are not significantly different at P≤ 0.05. Treated Control
Fig. 14:- Effect of different concentrations of T. harzianum inocula on number of galls on tomato plant roots
4.3.2. Experiment 2
4.3.2.1. Effect of different concentrations of the fungus A. oligospora inocula on number of Lemon leaves:-
In both seasons the highest concentration rate of the fungus inoculums significantly resulted in higher number of leaves compared with the other two rates and the untreated control through out the season,(Table 9and10). In the first season, table(9) showed that, the medium dosage concentration gave better result than the lower dosage rate. In the second season there is no significant differences between the medium and the lower concentration but they are better than the control, (Table 2).
Table 9:- Effect of different concentrations of the fungus A. oligospora inocula on number of Lemon leaves, season (1):-
Treatments after 1 after 2 after 3 after 4 after 5 after 6 after 7 after 8 after 9 month month month month month month month month month spores/ml
105 13.67a 18.00a 25.33a 34.00a 43.00a 58.67a 75.33a 78.33a 81.67a
104 9.67 b 11.3b 16.3b 21.6b 30.3b 44.0b 49.0b 54.6b 56.67b
103 7.00bc 9.00bc 12.33c 20.6b 24.6bc 31.33c 38.0bc 44.6bc 46.33bc
Control 5.33 b 6.33 b 8.67 c 14.67c 19.6b 26.33c 29.00c 33.6b 35.33c
CV% 15.07 12.22 12.22 12.17 15.26 17.49 14.38 11.88 11.27
SE± 0.78 0.79 1.11 1.60 2.59 4.05 3.44 3.63 3.58
Means in the same column followed by the same letter are not significantly different at P≤ 0.05.
Table 10:- Effect of different concentrations of the fungus A. oligospora inoculums on number of Lemon leaves, season 2:- Treatments after 1 after after after 4 after after 6 month 2month 3month month 5month month spores/ml
105 12.67 a 19.33 a 23.67 a 29.33 a 43.33a 53.67 a
104 7.67b 10.00 b 13.33b 21.33 b 30.00 b 38.00 b
103 6.67bc 8.67bc 11.00 b 20.33b 24.33 bc 29.67bc
Control 4.67c 5.67C 7.00c 13.00 c 15.00 c 19.67 c
CV% 13.15 14.40 13.05 8.25 17.10 14.92
SE± 0.60 0.91 1.04 1.00 2.78 3.04
Means in the same column followed by the same letter are not significantly different at P≤ 0.05. 0.001 ml
0.1 ml 0.01 ml Control
Fig. 15:- Effect of the different concentration of A. oligospora inocula on the vegetative growth of Lemon plants.
4.3.2.2. Effect of different concentrations of the fungus A. oligospora inocula on lemon stem length:-
In the first season all treatments significantly increased the stem length compared with the untreated control in the first month. Through out the season the highest concentration of the inoculums resulted in higher stem length compared with the medium and lower concentrations and the three concentrations significantly increased the stem length compared with the untreated control till the ninth month (Table 11 ).In the second season, (Table 12) indicated that, there is a difference in stem length but it is not significant in all treatments , however, there is significant difference between the highest concentration and the untreated control in up to the third month. At the fifth and sixth month the highest concentration of the inoculums significantly gave longer stem length than the other treatments. Table 11:- Effect of different concentrations of the fungus A. oligospora inocula on lemon stem length, season (1):- Treatments after 1 after 2 after 3 after 4 after 5 after 6 after 7 after 8 after 9 month month month month month month month month month spores/ml
105 11.50a 22.00a 28.33a 35.67a 45.00a 51.33a 55.67a 72.67a 75. 33a
104 12.75a 14.00b 20.00b 25.00b 28.67b 33.67b 42.67b 48.00b 49.67 b
103 10.00a 14.00b 17.33bc 20.00b 24.67b 27.33c 42.00b 45.67b 47.33b
Control 15.57b 08.33b 10.67 b 13.33c 15.00c 17.33d 23.ooc 25.00c 27.00 c
CV% 27.27 25.43 21.75 9.33 7.23 6.60 14.00 18.86 18.26
SE± 1.80 2.14 2.40 1.27 1.18 1.24 3.30 5.21 5.25
Table 12:- Effect of different concentrations of the fungus A. oligospora inocula on lemon stem length, season (2):-
Treatments after 1 after after after 4 after after 6 month 2month 3month month 5month month spores/ml
105 15.00 a 20.67a 26.33 a 30.33 a 35.33a 39.33 a
104 10.67 ab 15.00 ab 20.33ab 22.67ab 24.67b 27.00 b
103 9.00ab 14.33ab 15.33 bc 18.67bc 21.00 b 24.00b
Control 5.33 b 8.00 b 10.33 c 10.67b 13.00 c 15.00 c
CV% 32.10 27.13 16.82 25.09 9.61 11.82
SE± 1.85 2.27 1.76 2.98 1.31 1.81
Means in the same column followed by the same letter are not significantly different at P≤ 0.05 0.1 ml Control
Fig. 16:- Effect of the high concentration of A. oligospora inocula on the number of lemon leaves and stem length
4.3.2.3. Effect of different concentrations of the fungus A. oligospora inocula on lemon root length, fresh root weight and dry root weight:-
Table (13 ) showed that the higher concentration of the inoculums gave significantly the highest root length compared with the untreated control, also gave comparable result with the medium concentration and this last one resulted in comparable result with the lowest concentration. The three concentrations of the inoculums significantly resulted in high root fresh weight compared with the untreated control, however, they gave comparable result to each other.
Table 13:- Effect of different concentrations of the fungus A. oligospora inoculums on lemon root length, fresh root weight and dry root weight, season (1):-
Treatments Root Root fresh Root dry length weight weight spores/ml
105 61.33 a 14.67 a 5.52 a
104 55.33 ab 14.66 a 3.76 a
103 43.33 b 14.48 a 3.43 a
control 20.81 c 5.90 b 1.86 a
CV% 14.46 24.11 33.22
SE± 3.78 1.73 0.70
Means in the same column followed by the same letter are not significantly different at P≤ 0.05.
Table 14:- Effect of different concentrations of the fungus A. oligospora inocula on lemon root length, fresh root weight and dry root weight, season (2):-
Treatments Root length Root fresh Root dry weight weight spores/ml
105 66.67 a 18.28 a 7.07 a
104 52.33 b 13.73 b 5.23 b
103 31.83 c 11.75 b 2.80 c
Control 31.33 c 10.56 c 2.62 c CV% 22.71 37.41 18.61
SE± 5.97 2.93 0.48
Means in the same column followed by the same letter are not significantly different at P≤ 0.05.
4.3.2.4. Effect of the fungus inocula on number of nematode in the soil after removal of plant under test:-
In both seasons, Table (15 and 16 ) indicated that, higher number of nematodes had been obtained in the soil which is free from the inoculums compared with the othe r soil which treated with the fungus inoculums. The higher concentration gave the lowest number of nematodes compared with the other two concentrations.
Table 15:- Effect of the fungus A. oligospora inocula on number of nematodes in the soil after removal of plant under test, season (1) :-
Treatments Number of nematodes spores/ml
105 86.33 b
104 210.7 b
103 367.7 b
control 1411 a
CV% 69.65 SE± 23.46
Table 16:- Effect of the fungus inocula on number of nematodes in the soil after removal of plant under test:-
Treatments Number of nematodes spores/ml
105 114.00 b
104 181.7 b
103 304.3 b
control 901.7 a
CV% 45.67
SE± 98.99
4.3.3 Experiment 3 4.3.3.1. Effect of different concentrations of the fungus A. oligospora inocula on number of banana leaves:- In the first season and through out five months after transplanting banana, the plants which had been treated with the fungus inoculums plus the nematode gave higher number of leaves than those which were untreated with the fungus inoculums. The same results had been obtained in the second season, Table (17and18). Table 17:- Effect of different concentrations of the fungus A. oligospora inocula on number of banana leaves, season (1):- Treatments after 1 after after 3 after 4 after 5 month 2month month month month
Treated 8.00 10.00 12.67 14.00 15.67
Untreated 5.00 7.00 7.33 8.00 9.00
CV% 18.84 4.62 4.08 6.43 3.31
Table 18:- Effect of different concentrations of the fungus A. oligospora inoculums on number of banana leaves, season (2):-
Treatments after 1 after after after 4 after 5 month 2month 3month month month
Treated 7.00 9.66 11.67 13.00 14.33
Untreated 4.33 6.00 7.00 8.00 8.67
CV% 14.41 5.21 4.37 6.73
4.3.3.2. Effect of different concentrations of the fungus A. oligospora inocula on banana stem length:- In both seasons, higher stem length had been obtained in plants which treated with the fungus inoculums plus nematode compared with that one which was not treated till five months from transplanting, Table (19 and 20) Table 19:- Effect of different concentrations of the fungus A. oligospora inocula on banana stem length, season (1):-
Treatments after 1 after 2 after 3 after 4 after 5 month month month month month
Treated 11.00 15.00 19.67 27.33 31.00
Untreated 6.00 8.00 11.00 13.00 14.00
CV% 8.32 13.80 2.66 10.71 8.31
Table 20:- Effect of different concentrations of the fungus A. oligospora inocula on banana stem length, season 2:-
Treatments after 1 after after after 4 after 5 month 2month 3month month month
Treated 10.00 14.33 17.00 22.33 25.00
Untreated 5.33 7.00 9.67 11.5 13.33
CV% 5.32 7.65 6.12 5.23 4.19
Contol 0.001 ml 0.01 ml 0.1 ml
Fig. 17:- Effect of the different concentrations of A. oligospora inocula on the vegetative growth of banana plants.
4.3.3.3. Effect of different concentrations of the fungus A. oligospora inocula on banana fresh root weight and dry root weight:-
Table (21 and 22 ) indicated that, in both seasons, higher fresh root weight and higher dry root weight had been obtained in plants which had been treated with the fungus inoculums plus the nematode compared with the untreated one.
Table 21:- Effect of different concentrations of A. oligospora inocula on banana fresh root weight and dry root weight, season (1):-
Treatments Root fresh Root dry weight weight
Treated
85.09 10.34
Untreated
29.80 6.00
CV% 42.26 42.58
Table 22:- Effect of different concentrations of the fungus A. oligospora inocula on banana fresh root weight and dry root weight season (2):-
Treatments Root fresh Root dry weight weight
Treated 77.67 9.78
Untreated 29.85 6.41
CV% 39.64
Control 0.1 ml
Fig. 18:- Effect of the high concentration of A. oligospora inocula on the number of banana leaves and stem length
4.3.3.4. Effect of the fungus inoculums on number of nematode in the soil after removal of plant under test:- In the first season, after the removal of the plant , the soil had been treated with the fungus inoculums plus the nematode resulted in lower number of the nematodes thus the untreated ones gave higher number of nematodes. The same result had been obtained in the second season, Table (23 and24 ).
Table 23:- Effect of the fungus inocula on number of nematode in the soil after removal of plant under test, season (1):-
Treatments Number of nematodes
Treated 21.00
Untreated 62.00
CV% 18.01
Table 24:- Effect of the fungus inoculums on number of nematode in the soil after removal of plant under test, season 2:-
Treatments Number of nematodes
Treated 21.67
Untreated 66.00
CV% 16.79 Control 0.1 ml
Fig 19:- Effect of the high concentration of A. oligospora inocula on banana root weight
4.4. Effects of T. harzianum metabolites on the nematode:- Table 25 cleared that, the different dilutions of the fungus metabolite gave comparable percentage of mortality (16.8%, 14.19% and 11.65%) after 24 hours, however they significantly resulted in high percentage of mortality compared with the untreated control (3.78%). The same result had been obtained after 48 and 72 hours, the heights rate of the fungus metabolite significantly increased the percentage of mortality (66.24%) compared with the lowest rate and the untreated control (42.93 and 8.87%) respectively.
Table 25:- Percentage of mortality
Meabolites Mortality% Mortality % Mortality % dilutions after24 hours after 48 hours after72 hours
10-1 16.08 a 49.56 a 66.24 a
10-2 14.19 a 38.86 ab 52.11 ab
10-3 11.65 a 29.69 b 42.93 b
Control 3.78 b 4.98 c 8.87 c
CV% 16.32 18.41 19.04
SE± 3.81 3.69 4.77
Means in the same column followed by the same letter are not significantly different at P≤ 0.05.
CHAPTER FIVE
DISCUSSION
The study showed that, nematophagous fungi are specialized in trapping and digesting nematodes. Many ways of attacking nematode by fungi had been observed in this study, such as trapping by adhesive nets, adhesive knops, constricting rings and non-constricting rings, thus this is very important for using those fungi as biological control agent, these observations agreed with that of (Dackman, et al., 1992; Dijksterhuis, et al. 1994; Nordbring-Hertz, 1988; Nordbring-Hertz, et al., 1981; Tunlid, et al., 1992) who studied the relationships of Arthrobotrys oligospora with nematode and they studied the recognition mechanism of fungal infection. Also various aspects of mode of infection have been intensively studied by them. One way of trapping which had been noticed is that, fungi which had been trapped with adhesive nets, after trapping the nematode struggle and attached to another net and it attached to several nets. This finding is in agreement with Nordbring – Herttz (1972) and (Barron, 1977; Gray, 1988), who found that adhesive nets are common trapping devices of predacious fungi which are usually found in the Class Deuteromycetes which had been studied intensively and their mechanism of infection of nematodes and their efficacy in reducing nematode densities had been determined. Also nematode trapped by adhesive knobs had been noticed, this adhesive knob is morphologically distinct cell and it is considered to be highly specialized trapping devices. The same finding was obtained by (Gray, 1988) who reported that, adhesive knops are morphologically distinct cells, and they are considered to be highly specialized trapping devices. These knobs are covered by a thin layer of adhesive materials and either sessile or borne at the apex of a short non-adhesive stalk. When a nematode touches the knob, a flattened mass of adhesive is produced at the point of attachment, forming a thick pad. This increases the area of attachment many folds, insuring that the nematode is firmly held. Wimple and Young, (1984) reported that, adhesive knobs contain numerous electron – dense spherical bodies close to the part of the cell wall where is likely to come into contact with the nematode. In (Fig. 5) mycelium of Arthrobotrys inside the dead body of the nematode had been seen and also (in Fig.6) the mycelium and the spores of the fungus had been noticed. (Fig. 7) showed the mycelium inside the host body and all the body content had been consumed. All these findings are in agreement with the finding of (Barron, 1977), who found that in some fungi in Deuteromycetes, such as Arthrobotrys botryospora, Dactylaria psychrophila, and Arthrobotrys superb, also can capture nematodes with adhesive hyphae under certain conditions. These fungi produce adhesive materials that are deposited on some points of hyphal surfaces and when a nematode touches these points, it is captured. The hyphae produce appressoria that penetrate through the nematode body. After consuming all of the nematode content, the fungus draws all plasma back outside of the nematode body for the development of sporangia and spores. During this investigation it was noticed that as shown in Fig. 8 and 9 nematodes on a agar plate were seen dead with the toxin of the fungus Trichoderma. This finding agree with (Filtters, et al., 1992), who reported that, there are few studies have been done to characterize toxic compounds by fungi. Paecilomyces lilacinus releases chitinase and protease, which can cause undifferentiated Meloidogyne hapla eggs to become deformed and vaculate. Also our finding agree with (Mian et al. , 1982; Rodriguez-Kabana, et al. , 1989), who said that, fungi that produce antibiotic substances may be common in soil and more soil fungi that are antagonistic to nematodes through the release of toxins, antibiotics or enzymes remain to be discovered. Also Meyer et al., (2000) reported that non-enzymic factors produced by Tricoderma virens (syn. Gliocladium virens) inhibited Meloidogynye incognita egg hatch. Toxins produced by Fusarium spp. were tested on Meloidogyne incognita and some were highly toxic to the nematode (Ciancio, et al., 1988), this technique agree with that one which had been seen in Fig. 13, number of galls had been observed on the root of tomato plant which had been grown on soil affected with Meloidogyne compared with other tomato plant free from galls, this last one had been grown on soil containing the nematode Meloidogyne and the fungus Trichoderma, this means that the toxin which had been produced by the fungus Trichoderma killed the nematode. In this study it was found that, the high dosage of the metabolites of the fungus Trichoderma significantly increased the percentage of the nematode mortality after 24, 48 and 72 hours compared with the other rates and the untreated control, this finding is in agreement with that one studied by Balan and Gerber (1972) who studied the inactivation of the nematode by the fungus. The workers showed that filtrates of broths from fungal cultures had nematicidal activity. The filtrates caused complete and irreversible immobilization and death of the nematode within 24-48 hours. In this study the soil samples for search for the nematophagous fungi were collected from a depth ranging between 10cm down to a maximum of 35cm, it was noticed that the net forming endoparasites and the fungi which produce toxic material are isolated from soil collected from different depthes (10cm – 35cm). These findings agree with that one of (Gray and Baily,1985) who surveyed the distribution of nematophagous fungi in a deciduous woodland. They found that, nematophagous fungi were isolated throughout the soil profile down to maximum of 35cm deep. The net forming predators and endoparasites were isolated throughout the soil profile at all depths, although they were more abundant in the deeper rich soil. During this study, soil collected from different localities had been sprinkled on Petri-dishes containing corn meal agar and examined periodically. During the examination many ways of trapping had been noticed, one of them is a nematode captured by net as seen in (Fig.1). The fungus which trapped the nematode isolated and identified as Arthobotrys oligospora thus we can confirm that the nematophagous fungus A. oligospora is very common in Gezira and also we can say nematophagous fungi occur where ever nematodes can live. These findings are in agreement with (Gray,1987) who reported that Nematophagous fungi are world wide in distribution and have been reported from many countries. A. oligospora is recorded in many countries, Britain and Ireland (Gray, 1983). Also Leu et al., 1993 reported that the predacious fungi that were the most widely distributed in China include Monacrosporium thaumasium, M. eudermatum, A. oligospora, A. conoides, A. robusta, A. oviformis, and A. brochopaga. M. thaumasium was frequently encountered in the temperate areas in China. Also A. oligospora was most abundant in temperate soils in some surveys but not in other studies (Gray, 1983; Liu, et al. ,1993). Some observations had been noticed from this study in glasshouse experiment, that lower numbers of nematodes had been obtained when certain number of the nematode added to the soil which had been already affected with the fungus Trichoderma compared with the untreated control (soil free from the fungus inoculums). Other results obtained in laboratory experiment that Trichoderma metabolites increased the percentage of mortality (66.24) compared with the untreated control (8.87%). It could be said that the laboratory experiments is so much better than the glass house experiments, this results agree with that one of Chen et.al, (1996b) who reported that, some fungi are highly pathogenic to nematodes in the laboratory, but poor soil competitors. A. dactyloides is a predacious fungus forming constricting rings and also able to colonize eggs of H. glycine. The fungus is ubiquitous in distribution but a poor competitor in soil. Similarly, H. rhossiliensis can grow and sporulate well in artificial medium, but the fungus seems to be a poor competitor in natural field soil (Jaffee and zehr,1985). In the first season, (Table 1) showed that, the different concentrations of the fungus inoculums significantly increased the number of leaves from the first month of the observation up to the six month compared to the untreated control. In the second season, the different concentrations of the fungus inoculums significantly increased the number of leaves through out the season from the first month up to the six month as shown in (Table 2).All treatments significantly increased tomato stem length compared to the untreated control up to the six observation in the first season, (Table 3). In the second season, the high concentration of the fungus inoculums significantly increased the stem length compared with the medium and lower dosage rates of the inoculums and the untreated control, (Table 4).In the first season, the high concentration of the fungus inoculums (0.1) gave significantly (p≤ 0.05) the highest root length over the other two rates (0.01, 0.001) and the control. Also it gave significantly the highest mean root fresh and root dry weight over the control and the other two rates, (Table 5 ). In the second season, also the high concentration of the fungus inoculums gave significantly (p ≤0,05) the highest root length over the third concentration (0.001) and the control. This good growth in the vegetative part of plant throughout the two seasons indicated that, the inoculums of the fungus Trichoderma harzianum used during this study showed excellent and active effect against the root knot nematode (Meloidogyne Javanica) and there was evidence of biological control of this nematode by Trichoderma. Our findings agreed with that one of Budge and Whipps.1991; Chet,1987; Chet et al.,1997; Cole and Z. venyika, 1988; Lewis and Papavisaz,1984). Who said that, Trichoderma species were reported to be highly effective in controlling soil- borne pathogens. As nematodes are soil –borne pathogens Trichoderma sp. is good biocontrol agent for controlling nematodes. Fungal antagonists of nematode can be grouped into, (1) predacious fungi; (2) endoparasites of vermiform nematodes; (3) parasites of sedentary females and eggs; (4) fungi producing antibiotic substances; and (5) vesicular-arbuscular mycorrhizal (VAM) fungi. Fungi producing antibiotic substances is one of those fungal antagonists of nematode. In the tomato glass house experiment, when a certain number of the nematode Meloidogyne sp. added to the soil which had been mixed with the inoculums of different concentrations of the fungus Trichoderma, good results had been obtained concerning the vegetative growth of the tomato plant (stem length, number of leaves, root dry and fresh weight and shoot dry and fresh weight) also the tomato roots were healthy and free from root knot nematodes compared with the untreated control. Also, In both seasons number of nematodes which had been added to the soil with the different concentrations of the fungus Trichoderma inoculums before transplanting tomato plants significantly reduced compared with the untreated control after the removal of the plant at the end of the experiment (Tables7 and 8). The same results had been obtained in lemon glass house experiment that, the number of leaves ,stem length, root and shoot fresh weight and root and shoot dry weight were significantly better than the untreated control. Also in both seasons, (Tables 15and16 ) indicated that, higher number of nematodes had been obtained in the soil which is free from the inoculums compared with the other soil which treated with the fungus inoculums. The higher concentration gave the lowest number of nematodes compared with the other two concentrations. Those results agree with that one of Linford, (1937) and Linford, et al., (1938) who reported that, nematode antagonists have been observed in a wide range of organisms including fungi, bacteria, viruses, mites, insects and nematodes. Among those, fungal antagonists have been much important in regulating nematode population in soil. Also fungal antagonists have been studied since the first observation of nematophagous habit of the fungus, H. anguillulae, by Lohde in 1847. These results also agree with the report of (Kerry,1993; Stirling, 1991), who reported that, the interest in biological control occurred in the mid-1970s and this resulted from both the continuing environmental problems associated with the use of nematicides and demonstrations of suppressions of nematode by fungal parasites. Recently, more and more evidence showed that biological control of plant parasitic nematodes with fungal antagonistic is promising. In my knowledge I think that the nematophagous fungus A. oligospora has been tested for its potential as a biological control agent and shown to suppress nematode population densities and improve vegetative growth of banana and resulted in successful control of nematode.
CONCLUSION
1- In this study, many types of trapping by nematophagous fungi in soil collected randomly from different places grown with tomato, banana and lemon had been found.
2- The nematode Xephenema sp. which attack lemon trees had been seen captured by a net made by the mycelium of the fungus A. oligospora and it had been held at least at two points. The same nematode had bee captured by adhesive knob.
3- Another way of trapping that, the mycilium of the fungus A. oligospora had been seen inside the body of the nematode and consumed all the contents of it.
4- The fungus Trichoderma harzianum has been tested for its potential as a biological control agent and was found to suppress nematode population densities when it was added in different concentrations to 50 number of Meloidojyne javanica nematodes in soil planted with tomato. The fungus reduced the numbers of nematode galls on tomato roots compared with the untreated treated control and
5- resulted in excellent plant vegetative growth.
6- Growing of lemon plants on soil artificially infested with Xephenema sp. nematodes and treated with different concentrations of the fungus A. oligospora inocula significantly increased the number of lemon plant leaves, stem length, root length and also increased the fresh and dry root weight compared to the same soil without fungus inoculation. 7- Also the different concentrations of the fungus A. oligospora inocula resulted in excellent vegetative growth of banana plants when grown in soil artificially infested with the nematode R. similis.
8- Effect of the different rates of the fungus Trichoderma harzianum metabolites on the nematode Xiphenema sp. after 24 hours gave comparable percentage of mortality, 16.8%, 14.19% and 11.65% respectively, but significantly better than the untreated control that resulted in 3.78% mortality. After 72 hours, the highest concentrations of the fungal metabolites resulted in significantly high nematodes mortality (66.24%) compared with the lowest one (42 93%) and the untreated control (8.87%).
9- The rise in prices and resistance of organisms and pest to pesticides necessitates the search for other suitable way in controlling plant diseases.
10- Use of nematophagous fungi as biocontrol agents help in decreasing the cost of nematicides and reduce the application rates, thus conserving the ecosystem.
Recommendations:-
Due to the high prices of chemical pesticides and their hazards to the environment and unforeseen effects on human and animal health. It shoud be recommended that:-
1- More attention should be given to biological control, specially by nematophagous fungi. 2- It should be team work and in large scale. 3- Nematophagous fungi are easly to be isolated on culture media and used to inoculate soils cropped to plants susceptable to nematode diseases. 4- Fungal metabolites can also be used to inoculate soils to decrease infestation by variety of plant pathogens, specially soil borne diseases.
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