Hebron University Research Journal. H.U.R.J. is available online at Vol.(3), No.(1), pp.(1 – 15), 2007 http://www.hebron.edu/journal Biological Control of Rhizoctonia solani by Indigenous Trichoderma spp. Isolates from Palestine 1 1 *Radwan M. Barakat , Fadel Al-Mahareeq , 2 1 Mohammed S. Ali -Shtayeh , and Mohammad I. AL- Masri 1Faculty of Agriculture, Hebron University, Hebron- Palestine 2Faculty of Science, An-Najah National University, Nablus- Palestine Abstract: The effect of indigenous Trichoderma isolates against the soil-borne phytopathogen Rhizoctonia solani was investigated in dual culture and bioassay on bean plants. Ap- plication of the bioagent isolates as a conidial suspension (3*107) greatly reduced the disease index of bean plants caused by R. solani in different rates and the most effective Trichoderma harzianum isolate (Jn14) reduced the disease by 65%. In dual culture, the T. harzianum (Jn14) overgrew the pathogen R. solani in an average of 16.75 mm/day at 30 °C. In addition, the results showed that T. harzianum (Jn14) and T. hamatum (T36) were the most effective isolates at 25°C and inhibited R. solani mycelial growth by 42% and 78% respectively, due to fungitoxic metabolites production. The Effect of Trichoderma on bean seedlings growth was obvious; height was nearly doubled (160% - 200%), while fresh and dry weights increased by 133% and 217%, respectively. Ger- mination of bean seeds in treated soil with Trichoderma isolates occurred about four days earlier than those in untreated soils. The results revealed however some variation between isolates which was due to genetic variation, mycelium-coiling rate, sporulation rate, fungitoxic metabolites, induced growth response and temperature effect. Key words: Biological control, Damping off, Trichoderma, Rhizoctonia solani, Bean. * Corresponding author: [email protected] Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 املكافحة احليوية ملر�ض عفن اجلذور الريزوكتوين على نباتات الفا�سوليا با�ستخدام عزﻻت فل�سطينية من فطر الرتيكوديرما امللخ�ض: خﻻل هذه الدرا�سة مت فح�ض مقدرة 47 عزلة حملية من فطر Trichoderma على مكافحة مر�ض عفن اجلذور الريزوكتوين )Rhizoctonia solani( با�ستخدام طريقة التداخل الفطري )Dual Culture(ومعاملة نباتات 7 الفا�سوليا الكاملة)Bioassay(. أاظهرت نتائج اإ�سافة العزﻻت كمعلق يحتوي ابواغ الفطر برتكيز ) g soil/3x10( اىل الرتبة ملكافحة املر�ض على نباتات الفا�سوليا أن اهناك تباين معنوي يف مكافحة املر�ض حيث أادت أاقوى العزﻻت )Jn14(اىل تقليل �سدة اﻻ�سابة باملر�ض بن�سبة 65 %. أاظهرت نتائج درا�سة التداخل الفطري بني عزﻻت فطر الرتيكوديرما وفطر R. solani أان هناك تباين معنوي بني العزﻻت يف التطفل على الفطر املمر�ض وكان معدل منو اقوى عزلة يف التطفل 16.75 ملم / يوم. ووجد من نتائج درا�سة ت أثريا احلرارة على عملية التداخل بني أاقوى العزﻻت والفطر املمر�ض بان العزلة )Jn14( كانت أاقواها، و أاف�سل درجات احلرارة لنموها 25-30 درجة مئوية. وعند درا�سة آالية عمل فطر الرتيكوديرما على الفطر املمر�ض؛ وجد أان فطر الرتيكوديرما لديه املقدرة على التطفل على الفطر املمر�ض واإنتاج املثبطات الفطرية عند تربيته على بيئة البطاطا املغذية ال�سائلة )PDB(. و أاظهرت النتائج بان املثبطات املنتجة من العزلتني )Jn14 and T36( عند مزجها بن�سبة 10 % مع بيئة البطاطا املغذية ال�سلبة PDA قد قللت معدل منو الفطر املمر�ض بن�سبة 42 % و 78 % )على التوايل( على درجة حرارة 25م°. كما وجد أان فطر الرتيكوديرما يعمل على تبكري انبات بذور نباتات الفا�سوليا املعاملة مبعدل 4 أايام من البذور الغري معاملة واىل زيادة طول النباتات بن�سبة 160%-200% وزيادة وزن النباتات بن�سبة 133%- . 217%وعزي التباين بني العزﻻت يف مكافحة املر�ض اىل مقدرتها على التطفل الفطري، �رسعة منوها، انتاج اﻻبواغ، درجة احلرارة ومقدرتها على انتاج املواد املثبطة، اإ�س ًافة اىل اﻻختﻻف الوراثي واجليني بني العزﻻت. Introduction: Damping off caused by Rhizoctonia impact on the environment and has been solani is an economically important dis- recently phased out due to the public ease on beans in Palestinian agriculture. concern and international agreements. Different fungicides and soil fumigants The use of antagonistic microorgan- are currently used to control soilborne isms against R. solani has been inves- plant pathogens including R. solani. tigated as one of the alternative control However, many of these compounds methods. Trichoderma harzianum is proved to be quite toxic to the environ- well documented as effective biologi- ment and to the ground water. Methyl cal agents for R. solani control in soil bromide is a good example for a very (Papavizas, 1985, Coley-Smith et al., efficient soil fumigant that has a great 1991, Samuels, 1996, Prasun and Kan- Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 thadai, 1997, and Sivasithamparam and and lysis of pathogen and /or compe- Ghisalberti, 1998, and Howell, 2003). tition for limiting factors in the rhizo- Biological control can be specific and sphere mainly iron and carbon (Chet, depends on soil, environmental factors, 1990). Another mechanism has been location, and season in addition to crop suggested by kleifeld and Chet (1992) and pathogen. The biological control of and related to Trichoderma-induced re- the pathogen on bean plants by using sistance in host plants to fungal attack. Trichoderma spp. and/or combination The increased growth response (IGR) with other techniques has been inves- of plants depends however, on the abil- tigated. Application of small non-effec- ity of the Trichoderma to survive, de- tive doses (1-2 µg/kg) of Pentachloron- velop in the rhizosphere, and varies as itrobenzene (PCNB) to soil along with well with the substrate (Kleifeld and a Trichoderma preparation (2g/kg), de- Chet, 1992, and Yedidia et al., 1999). creased the incidence of eggplant damp- In addition, Harman (2000) established ing off caused by R. solani from 13 to that Trichoderma spp. are opportunistic 40%, while T. harzianum alone reduced plant colonizers that affect plant growth disease incidence by 26% (Hadar et al. by promoting abundant and healthy ,1979). The combination of sublethal plant roots, possibly via the produc- heat treatment dose and T. harzianum, tion or control of plant hormones. In- enhanced control of disease on beans creased growth response has been dem- (by 90 to 100%) under greenhouse con- onstrated by several other investigators ditions (Elad et al., 1980). Aziz et al. (Altomare et al., 1999; Anusuya and (1997) found that the application of a Jayarajan, 1998). They demonstrated wheat bran preparation of Trichoderma the ability of T. viride and T. harzianum lignorum conidia (8×106 conidia/seed) to solubilize insoluble tricalcium phos- at a rate of 15 and 20 g/500 g soil de- phate in vitro. creased the damping-off percentage by The objective of this study was to 12% and 6%, respectively, as compared evaluate the potential of the bio-agent to control. In addition, they found that Trichoderma isolates recovered from the application of wheat bran prepara- Palestinian agricultural fields in cont- tion of Trichoderma lignorum (5 × 106 trolling the soil-borne phytopathogen cfu/g) at a rate of 2.5 g /500 g of soil de- Rhizoctonia solani. creased the damping-off percentage by 45%. Furthermore, Noble and Coven- Materials and methods try (2005) showed that the combination of Trichoderma harzianum and organic Trichoderma isolates: amendment can be used to control soil- Forty-seven native isolates of Trichode- borne pathogens including R. solani. rma were recovered previously from The ability of Trichoderma to reduce various agricultural soils in the West the disease is well known and related Bank; the antagonistic potential against to the antagonistic properties of Tri- Botrytis cinerea and Sclerotium rolfsii choderma, which involve parasitism in dual culture and bioassay was inten- Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 sively studied (Barakat and Al-Masri, potato dextrose agar (PDA) at 25°C un- 2005, Barakat et al., 2006). These iso- der continuous fluorescent light. After lates were used in this study to evalu- 7-10 days of incubation, conidia were ate their biocontrol potential against R. harvested from cultures by flooding solani damping off of bean. the plates with 10 ml of sterile distilled water and removed by sterile bent glass rod and poured into sterile test tubes Preparation of Rhizoctonia solani and agitated for 15 sec. with vibrating inoculum: agitator. The resulting suspensions were filtered through a layer of sterile tissue The R. solani isolate (Rh1) used in the papers and the conidial concentration in study was originally recovered from the suspension was determined with a diseased bean plants (2000) and clas- haemacytometer. Sterile distilled water sified under antastomonsis group 1 act- was added to bring the concentration to cording to (Carling, 1996). Fifty mil- 3*107 conidia / ml (Mihuta & Rowe, liliters of potato dextrose broth (PDB) 1986). Four milliliters of the suspen- in 250 ml Erlenmeyer flasks was inocut- sion (3*107 conidia / ml) was added to lated with two 7mm-diameter mycelial pots containing a 0.5-kg sand soil pre- disks from 7- days- old PDA cultures of viously autoclaved at 121°C for 1 hr on R. solani and incubated at 27°C for 10 three successive days. days. After incubation, the upper solid The Trichoderma-inoculated soils were layers of fungal growth were washed incubated for seven days at 25 °C and and air-dried with tissue paper layers. then mixed thoroughly with the 0.9 g The amount needed from this prepara- R. solani preparation. Each plastic pot tion was (0.9 g dry preparation of R. (10 cm diameter) was filled with nont solani / 0.5 kg soil). The air-dry myc- autoclaved sand to 2/3 of pot volume elium was placed in autoclaved 250ml and then seeded with six bean seeds Erlenmeyer flask, shredded by Ultra (Phaseolus vulgaris).