Biological Control of Rhizoctonia Solani by Indigenous Trichoderma Spp

Home , Rhizoctonia solani, Trichoderma viride

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. App- 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-m 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�صوليا با�ستخدام عزالت فل�سطينية من فطر الرتيكوديرما

امللخ�ص:

خالل هذه الدرا�سة مت فح�ص 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�صوليا الكاملة)Bioassay(. أ�ظهرت نتائج �إ�ضافة العزالت كمعلق يحتوي ابواغ الفطر برتكيز ) g soil/3x10( اىل الرتبة ملكافحة املر�ض على نباتات الفا�صوليا أن �هناك تباين معنوي يف مكافحة املر�ض حيث أ�دت أ�قوى العزالت )Jn14(اىل تقليل �شدة اال�صابة باملر�ض بن�سبة 65 %. أ�ظهرت نتائج درا�سة التداخل الفطري بني عزالت فطر الرتيكوديرما وفطر R. solani أ�ن هناك تباين معنوي بني العزالت يف التطفل على الفطر املمر�ض وكان معدل منو اقوى عزلة يف التطفل 16.75 ملم / يوم. ووجد من نتائج درا�سة ت أثري� احلرارة على عملية التداخل بني أ�قوى العزالت والفطر املمر�ض بان العزلة )Jn14( كانت أ�قواها، و أ�ف�ضل درجات احلرارة لنموها 25-30 درجة مئوية. وعند درا�سة آ�لية عمل فطر الرتيكوديرما على الفطر املمر�ض؛ وجد أ�ن فطر الرتيكوديرما لديه املقدرة على التطفل على الفطر املمر�ض و�إنتاج املثبطات الفطرية عند تربيته على بيئة البطاطا املغذية ال�سائلة )PDB(. و أ�ظهرت النتائج بان املثبطات املنتجة من العزلتني )Jn14 and T36( عند مزجها بن�سبة 10 % مع بيئة البطاطا املغذية ال�صلبة PDA قد قللت معدل منو الفطر املمر�ض بن�سبة 42 % و 78 % )على التوايل( على درجة حرارة 25م°. كما وجد أ�ن فطر الرتيكوديرما يعمل على تبكري انبات بذور نباتات الفا�صوليا املعاملة مبعدل 4 أ�يام من البذور الغري معاملة واىل زيادة طول النباتات بن�سبة 160%-200% وزيادة وزن النباتات بن�سبة 133%- . 217%وعزي التباين بني العزالت يف مكافحة املر�ض اىل مقدرتها على التطفل الفطري، �رسعة منوها، انتاج االبواغ، درجة احلرارة ومقدرتها على انتاج املواد املثبطة، �إ�ض ًافةاىل االختالف الوراثي واجليني بني العزالت.

Introduction:

Damping off caused by Rhizoctonia impact on the environment and has been solani is an economically important dis-e 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-i are currently used to control soilborne isms against R. solani has been invest- 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-m well documented as effective biologi-c 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 Kant- 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 compet- Ghisalberti, 1998, and Howell, 2003). tition for limiting factors in the rhizos- 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-s 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-t of plants depends however, on the abili- tigated. Application of small non-effect- ity of the Trichoderma to survive, de-v tive doses (1-2 µg/kg) of Pentachloroni- 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-c Chet, 1992, and Yedidia et al., 1999). creased the incidence of eggplant damp-i 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 product- heat treatment dose and T. harzianum, tion or control of plant hormones. Inc- enhanced control of disease on beans creased growth response has been dem-o (by 90 to 100%) under greenhouse cond- 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 phosp- at a rate of 15 and 20 g/500 g soil de-c 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 preparat- 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 dec- Rhizoctonia solani. creased the damping-off percentage by 45%. Furthermore, Noble and Covent- Materials and methods try (2005) showed that the combination of Trichoderma harzianum and organic Trichoderma isolates: amendment can be used to control soilb- Forty-seven native isolates of Trichode-r 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-c Botrytis cinerea and Sclerotium rolfsii choderma, which involve parasitism in dual culture and bioassay was inten-s 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 und- 2005, Barakat et al., 2006). These isol- der continuous fluorescent light. After lates were used in this study to evalua- 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 class- haemacytometer. Sterile distilled water sified under antastomonsis group 1 acc- was added to bring the concentration to cording to (Carling, 1996). Fifty mil-l 3*107 conidia / ml (Mihuta & Rowe, liliters of potato dextrose broth (PDB) 1986). Four milliliters of the suspens- in 250 ml Erlenmeyer flasks was inocul- sion (3*107 conidia / ml) was added to lated with two 7mm-diameter mycelial pots containing a 0.5-kg sand soil prev- 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-t R. solani preparation. Each plastic pot tion was (0.9 g dry preparation of R. (10 cm diameter) was filled with non- solani / 0.5 kg soil). The air-dry myc-e 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). The final mixture thorax in distilled water and was then containing the pathogen and the bio-a passed through a 2 mm sieve to obtain agent was placed in the last 1/3 volume inoculum pieces of 1-2 mm in length. of pots which were previously seeded with bean seeds. The experimental de-s Evaluation of antagonistic potential sign was completely randomized with (Bioassay): five replicates (pots) for eachTrichode -r rma isolate. The soil of the control treatm- The screening for antagonistic poten-t ment contained the pathogen (R. solani tial was studied using the method of ) without the Trichoderma. Plants were Mihuta and Rowe (1986). Forty-seven harvested after three weeks from seed-i local isolates of Trichoderma were test-e ing and incubated in the growth chamb- ed in growth chamber for antagonism ber at 25°C under a 12-hr photoperiod. to R. solani . Isolates were grown on All seedlings were uprooted, and the Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 hypocotyls were evaluated for infection randomized design with five replicates by R. solani on a scale from 1 to 4: (1= (Petri dishes) used. The effect of tem-p symptomless, 2= small lesions with no perature on dual culture interaction bet- hypocotyl constriction, 3= large lesions tween the most effective Trichoderma with some hypocotyl constriction, and isolates (H2, J8, Jn14, Jn21, Q27, T33, 4= hypocotyl girdled). T36, N38 and R42) and R. solani was These ratings were converted to a dis-e repeated studied as described above und- ease index (DI) value for each pot by der different temperatures (10, 15, 25, using the formula: DI = 30 * (1A + 2B 30, 35 and 40°C) for 3-5 days in closed + 3C +4D) / N polyethylene bags lined with moist where A, B, C, and D represent the tissue papers (to prevent desiccation number of seedlings rated as 1, 2, 3, or of the media), and observed regularly 4 respectively; 30– the number of seeds for ability of the fungus to restrict the planted; and N– the number of seedl- growth, or to overgrow the other. The lings rated after three weeks. experimental design was completely randomized with four replicates (plates) for each treatment. Mode of Action A) Dual culture interaction: B) Hyphal interaction on thin film of Dual culture interaction between the agar: pathogen, R. solani and the Trichode-r rma isolates were studied using the This procedure was done according to following method (Dennis & Webster, that of Laing & Deacon (1991). Hyphal 1971 b and Prasun & Kanthadai, 1997): interaction was studied on sterile glass 5-mm diameter mycelial blocks (7- cover slips coated with 2% water agar days-old) cut from the margin of each (20g Difco agar / 1L distilled water). Trichoderma isolate and of the pathog- Each cover slip was immersed for 1-2 gen colonies grown on potato dextrose sec in autoclaved melted water agar at agar (PDA) were placed 3 cm apart on about 45 °C, allowed to drain and then the PDA surface. Five 90 mm diameter placed on the surface of 2% solidified Petri dishes were incubated at 25 °C water agar in a 90 mm diameter Petri under continuous light and inspected dishes, so that a thin film of agar set on daily for approximately 8-9 consecu-t the upper surface. Five mm disk of one- tive days for interaction. The fungus week-old growing colonies cut from colony margins would meet 2-3 days the margins of each of R. solani and after inoculation. The area of interac-t Trichoderma isolates were placed 3 cm tion of the bioagent and the pathogen apart on the agar surface and then incub- was measured every 24 hours after bated at 25°C. Cultures were inspected contact. The overgrowth rate (mm/day) daily for interaction; R. solani and Tri-c was measured during 10 days after cont- choderma colony margins made cont- tact. The experiment was completely tact across the coated coverslips in less Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 than three days. Each cover slip was fication. Plates were incubated at 15, removed carefully without damaging 20, 25, 30, and 35 °C; unamended PDA the mycelial contact and was inverted plates served as the control. The linear on a sterile microscopic slide (25.4 x growth rate of R. solani was measured 76.2). Microscopic examination was after 48 hour and the growth rate was carried out using fresh direct mounts in calculated as cm2 /day. The experimen-t Lactephenol cotton blue under medium tal design was completely randomized and high magnifications (20 and 40x), with four replicates (plates) for each respectively. Specimens were always treatment. sealed by nail varnish to prevent dry-i ing. Mycoparasitism was indicated by hyphal coiling and internal coloniza-t Plant’s increased growth response (IGR): tion of the host hyphae (R. solani) by the bioagent Trichoderma. The ability of Trichoderma to prot- tect the emergence of seedlings, and C) Production of toxic metabolites to improve plant’s growth was tested. (Antibiosis): Fifteen Trichoderma isolates (H2, H3, H4, J8, J9, Jn14, Jn18, Jn21, Q26, Q27, The ability of Trichoderma spp. to inh- T36, T37, N38, R42, and B47) were hibit the mycelial growth of R. solani used. The isolates were grown on Petri through production of fungitoxic met- plates (90-mm in diameter) containing tabolites at different temperatures was PDA for 10 days at 25°C under illumi-n tested according to the method men-t nation. Conidia were harvested from tioned by Dennis and Webster (1971 cultures, washed several times in wat- a and b). Fifty ml of potato dextrose ter, and suspended in 0.001% Tween broth (PDB), pH 6 in 250 ml Erlenmeye- 20 (Polyoxyethylenesorbitan, Sigma- er flasks were inoculated with 7mm- Aldrich. Com.) (Chang et al., 1986). agar disks from 7-day-old PDA cultures Conidial suspensions were added to the of three Trichoderma isolates J8, Jn14, peat moss growth substrate at the con-c 6 and T36 and incubated at 25°C without centration of 5x10 conidia per gram of shaking. After 10 days of incubation, soil and incubated for 14 days at 25°C. the cultures were filtered through a Mil-l After 14 days, six bean (Phaseolus vul-g lipore membrane filter (0.45 µm) and garis) seeds were sown per pot and five autoclaved at 121°C for 15 minutes. replicates were employed. Plants were The culture filtrate (1.2 ml) was placed grown in growth chamber at 25 2 °C for in Petri dishes (90-mm diameter) and 4 weeks. The various measurements of approximately 12 ml of PDA was added plant’s growth responses were made inc- and mixed with the filtrate (10% v/v). cluding number and time of emergence The filtrate-amended PDA plates were of seedlings. The emergence was evalua- then centrally inoculated with 7-mm ated after 3 and 7 days. Plant heights mycelial plugs of R. solani after solidi-f were measured from soil surface to Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 apical buds after 4 weeks. Concerni- control. The isolates (T34, N40, Jn13, ing fresh and dry weights, plants were B46, B44, B45, Jn22, and H1) were less washed under running tap water to re-m effective; they reduced disease index by move soil from roots; plants were then 1.5%-16.4% compared to the control. dried at 80 °C in drying oven after rec- cording fresh weights. After 72hr, plant Mode of action dry weights were determined (Shenker Dual culture interaction: et al., 1992). The ability of Trichoderma to inhibit Statistical analysis: the mycelial growth of R. solani in dual culture was determined on PDA Data of all experiments were analyzed medium (Fig. 2). A clear zone of intera- statistically using One-Way Analysis of action was formed on PDA media at the Variance (ANOVA) to test for signific- point of contact between the mycelium cance; Fisher test was used for mean of R. solani and Trichoderma. Results separations by SigmaStat Software of interaction between the Trichoderma (1999). isolates mycelia and R. solani mycelia showed statistically significant differe- Results: ences in the overgrowth rate between the different isolates (LSD= 0.867; Evaluation of antagonistic potential in P≤0.05) (Fig. 2). Means of overgrowth bioassay: rate (mm/day) of Trichoderma isolates Trichoderma isolates reduced disease on R. solani ranged from 2.4 for the index of bean plants caused by R. Trichoderma isolate H5 to 9.9 for the solani in different rates (LSD= 19.588; Trichoderma isolate Jn17 (Trichode-r P≤0.05); means of disease index ranged rma atroviride). Results showed that from 38.2 to 111 (Fig. 1). Thirty nine the most effective Trichoderma isolates Trichoderma isolates (Jn14, R42, Jn21, against R. solani were (Jn17, J8, J9, T33, T36, H2, N38, J8, Q28, J9, H3, T36, Jn18, Jn14, Jn21, and H1). H4, T37, N39, Q26, Jn17, Jn18, B47, The effect of different temperatures on Q24, Q29, J10, Q27, Q30, R43, Jn20, the interaction between the most effec-t Q25, Jn11, Jn19, Jn23, H7, T35, T31, tive Trichoderma isolates mycelia and R41, H5, T32, H6, Jn15, Jn12, and the R. solani mycelia was evaluated. Jn16), significantly reduced disease ind- Results showed significant differences dex by 18.2%- 65.6%, compared to the between isolates (LSD= 1.635; P ≤0.05) control. Disease index was reduced on in this respect (Fig.3). The interactions bean seedlings by 65.6%, 60.5%, 56%, between the two fungi were highly 54.9%, 54.8%, and 53.4%, respectively, dependent on temperature. The mean when the most effective Trichoderma overgrowth rate ranged from 0.02 mm/ isolates (Jn14, R42, Jn21, T33, T36, day for the isolate Q28 (Trichoderma and H2) were used compared to the hamatum) at 15 °C and 16.75 mm/day Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 at 30 °C for the isolate Jn14 (Trichode-r duced significantly in amended media rma harzianum) (Fig. 3). at 20 °C and 25 °C (LSD = 0.942; P ≤0.05) (Fig. 4). The variations between Hyphal interaction on thin film of agar: isolates were obvious at 25 °C. R. solani growth rate inhibition percentages were The Trichoderma isolates (H2, J8, Jn14, 42%, 78%, and 50.5% when growth Jn17, Jn21, T33 and T36) showed ident- media were amended with 10% of PDB tical mode of action during interacting containing metabolites produced by the with R. solani on water agar films. Aft- isolates T36, Jn14, and J8, respectively. ter contact, the hyphae of Trichoderma Results showed that Jn14 metabolites grew along the pathogen hyphae; somet- was the most effective at all tempera-t times the main hyphae coiled around tures (15 °C, 20°C, and 25 °C), and the host or produced short branches reduced R. solani mycelial growth by that tightly surrounded the host hyphae. the percentages of 76%, 66%, and 78%, Dense coiling around host hyphae and respectively (Fig. 4). internal growth within the host myc-e Plant’s increased growth response elium had commonly been seen duri- (IGR): ing interaction between R. solani and Trichoderma; disintegration of the host The results showed that there was a cell wall was observed as well. significant increase for each of the par- Microscopic examination had also rameters measured (plant’s emergence, revealed that the isolate Jn14 (T. har-z heights, and fresh and dry weights) zianum) was very efficient in coiling in bean seedlings, 4 weeks after sowi- and interned colonization of R. solani ing compared to the non-treated seedl- hyphae. Coiling of T. harzianum (Jn14), lings (Table 1). Bean seedlings treated T. hamatum (T36), and T. Pseudokoni- with Trichoderma isolates J8 and T36 ingii (Jn21) hyphae around R. solani increased in height by 160 to 200%, hyphae was noticed on 40x microscop-i respectively. In addition, seedlings ic magnification. Host hyphae internal treated with the isolates Jn14 and Jn21 colonization by the Trichoderma isol- increased in fresh weights in the range lates Jn14, T33, and J8 was very clear of 133 % to 217%, respectively. There and obvious. The types of hyphal int- were no significant differences, howe- teractions were observed in this study; ever, in respect to plant dry weights coiling around the host hyphae; pene- within treatments. Germination of bean etration of the host hyphae and lack of seeds planted in soils treated with Tri-c cytoplasm in host cells; and subsequent choderma isolates mentioned above lysis of the infected hyphae. started about four days earlier than those planted in untreated soil. Germi-n Production of toxic metabolites nation of seeds planted in Trichoderma (Antibiosis): (Jn 14 and T36) treated soils increased The growth rate of R. solani was re-d in the range of (16.7% - 55.6%), com-p pared to the control. Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007

140 130 120 110 100 x e

d 90 n i

e 80 s a

e 70 s i

D 60 50 40 30 20 10 0 0 1 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 8 9 0 1 2 3 4 5 6 7 1 2 3 4 5 6 7 J J 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 K H H H H H H H K J n n n n n n n n n n n n n T T T T T T T B B B B N N N R R R Q Q Q Q Q Q Q C J J J J J J J J J J J J J C Figure 1. The effect of Trichoderma isolates on disease index caused by R. solani on bean plants (LSD=19.6).

12 ) y a d /

m 10 m ( e t a r 8 h t w o r g r 6 e v o m u i

l 4 e c y M 2

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 J J 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 H H H H H H H J n n n n n n n n n n n n n T T T T T T T B B B B N N N R R R Q Q Q Q Q Q Q J J J J J J J J J J J J J

Figure 2. Mycelium overgrowth rate (mm/day) of local Trichoderma isolates over Rhizoctonia solani in dual PDA culture incubated at 25 ºC (LSD=0.86).

2 Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 10

20 20 H2

) 18

) 18 1 J8 - 1 J8 - 16 d 16 d Jn14

m 14

m Jn21

m 12 Jn21

( 12 (

s 10 Q28 s Q28 e 10 e t t a 8 T33 a r 8 T33 r

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w 4 w 4 o N38 o r N38 r 2 g 2 g r

r R42

e R42

e 0

v 0

v 10 15 20 25 30 35 40

O 10 15 20 25 30 35 40 O

Temperature (ºC) Temperature (ºC) Figure 3. Overgrowth rates of Trichoderma isolates (H2, J8, Jn14, Jn21, Q28, T33, T36, N38, and R42) over R. solani growing on PDA medium in dual culture at dif-f ferent temperatures (10, 15, 25, 30, 35 and 40 0C) (LSD=1.635).

12.0 Ck J8 Jn14 T36 ) Ck J8 Jn14 T36 ) 1 - 1 - d d

2 10.0 2 10.0 m m m m ( (

e 8.0 e t 8.0 t a a r r h t h t

w 6.0

w 6.0 o o r r g g m m

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i 4.0 i l l e e c c y y 2.0 M 2.0 M

0.0 15 20 25 Temperature (ºC) Figure 4. Mycelium growth rate (mm2d-1) of R. solani growing on PDA medium amended with metabolites produced by the Trichoderma isolates (J8, Jn14, T36) and incubated at different temperatures (15, 20, and 25 ºC) (LSD=0.942).

3 Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 11

Table 1. The effect of Trichoderma isolates on bean plant’s growth

Emergence Plant height Plant weight (g) Isolate 3 days 7days Fresh Dry CK 0.00* c 3.6 b 18.3* c 5.9 c 0.59 c H2 1.6 c 4.6 a b 29.9 b 10.4 a b 1.0 b H3 1.5 c 4.75 a b 31.0 b 1.06 a b 1.07 a b H4 1.6 c 4.4 a b 34.7 a 11.1 a 1.11 a J8 1.75 c 4.25 a b 29.8 b 8.9 b 0.89 b J9 1.0 c 4.2 a b 29.8 b 8.8 b 0.88 b Jn14 3.4 b 5.6 a 36.7 a 12.9 a 1.28 a Jn18 2.0 c 4.2 a b 29.7 b 11.2 a 1.12 a b Jn21 1.8 c 5.2 a 32.1 b 8.4 b c 0.84 b Q27 1.6 c 4.8 a b 32.5 b 11.8 c 1.18 a b Q28 1.6 c 4.8 a b 33.4 ab 11.9 a 1.19 a b T36 2.2 c 5.6 a 36.8 a 12.7 a 1.27 a T37 0.6 c 4.6 a 29.7 b 9.5 b c 0.95 b N38 2.2 c 5.2 a 34.7 a 11.0 a 1.10 a b R42 2.4 b c 5.2 a 34.7 a 9.8 b c 0.98 b B47 1.2 c 4.6 a b 34.0 a 10.9 a b 1.09 a b LSD (P≤ 0.05) 0.998 4.19 2.23 0.22

* Mean of five replicates; values followed by the same letter within columns are not significantly different according to Fisher LSD test. gen and formed branches that coiled Discussion: around them. Dense coiling of the Tri-c choderma isolate (H2) around R. solani Results showed that the dual culture hyphae was observed. Light microsco-p contacts between Trichoderma isolates py revealed the penetration and growth and R. solani occurred after 2 days of of Trichoderma isolates (Jn14, T33, growth. All Trichoderma isolates grew and J8) inside the hyphae of R. solani . over the R. solani colonies and degraded Similar results were obtained earlier on its mycelium. The study further revealed Trichoderma harzianum (Jn14) against that the Trichoderma isolates (H2, J8, Botrytis cinerea(Barakat and Al-Masri, Jn14, Jn17, Jn21, T33 and T36) parasit-i 2005) and Sclerotium rolfsii (Barakat ized the hyphae of R. solani . Trichode-r et al., 2006). Other observations have rma hyphae of the isolates (Jn14, Jn21, been reported as well for Trichoderma and T36) grew over those of the patho-g harzianum and Sclerotinia sclerotiorum Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 12 interaction by Inbar et al. (1996). The ed earlier by 4 days than those planted antagonistic ability of Trichoderma iso-l in nontreated soils in addition to a bett- lates, however, is highly variable (Chet, ter emergence rate. In relation to this, 1990), as was shown in this study in Yedidia et al. (1999) suggested that a which only 11.54% of the Trichoderma 30% increase in cucumber seedling isolates tested were effective in control-l emergence was observed up to 8 days ling R. solani in the bioassay studies after sowing when soil was amended done in the growth Chamber. The most with T. harzianum propagules. This was effective isolates were (Jn14, Jn21, explained by (Kleifeld and Chet, 1992) T33, T36, H2, and R42). who referred to the ability of Trichode-r Application of Trichoderma as a con- rma to inhibit minor pathogens in the nidial suspension reduced disease in-d rhizosphere which might induce seed dex caused by R. solani by 65.6%. rots and preemergence damping off. The ability of Trichoderma to reduce Furthermore, seedlings grown in Tri-c diseases caused by soilborne pathogens choderma treated soils recorded higher is well known and related to the antagon- values of plant heights and weights. nistic properties of Trichoderma, which Some investigators reported that the inc- involve parasitism and lysis of patho-g creased growth response caused by Tri-c genic fungi and /or competition for lim-i choderma isolates resulted in large inc- iting factors in the rhizosphere mainly crease in the root area and root lengths iron and carbon (Sivan and Chet, 1986). and may be related to the effect on root Another mechanism has been suggested system. Yedidia et al. (1999) suggested by kleifeld and Chet (1992) and related a direct role for T. harzianum in mineral to Trichoderma-induced resistance in uptake by the plant at a very early stage host plants to fungal attack. Furtherm- of fungal-plant association. In addition, more, Aziz et al. (1997) reported the eff- Harman (2000) established that Tri-c fect of plant exudates on the inhibition choderma spp. are opportunistic plant of conidia germination in vitro and on colonizers that affect plant growth by the suppression of Rhizoctonia damp-i promoting abundant and healthy plant ing-off of bean in vivo when Trichode-r roots, possibly via the production or rma lignorum was applied. In the prese- control of plant hormones. ence of bean exudates, the reduction in The effect of temperature on the interac-t Rhizoctonia damping-off of bean by tion between the bioagent and the path-o Trichoderma lignorum was obvious. ogens was evaluated as well. Trichode-r Increased growth response of several rma isolates overgrew the pathogen (R. plants including vegetables, followi- solani ) in dual culture at the tempera-t ing the application of Trichoderma tures 15-35 oC. The overgrowth rate of to pathogen-free soil has been docum- Trichoderma isolate (Jn14) reached a mented (Baker, 1989; Chang et al., peak at the temperature of 30 oC. Howe- 1986; Kleifeld and Chet, 1992). In this ever, the mean overgrowth rate of Tri-c study, bean seeds which were planted choderma isolate (Jn14) reached a peak in Trichoderma treated soils germinat-e at 25 oC. Similar results were demon-s Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 13 strated by Prasun and Kanthdai (1997). 74, 464-466. In addition to possible mycoparasitism, 3. Aziz, N. H., El-Fouly, M. Z., El-Es-s results of this study revealed that the sawy, A. A., and Khalaf, M. A. (1997). growth rate of R. solani was reduced Influence of bean seedling root exudates due to the production of fungitoxic me-t on the rhizosphere colonization by Tri-c tabolites by Trichoderma isolates at diff- choderma lingorum for the control of ferent temperatures. The growth rate of Rhizoctonia solani. Botanical Bulletin R. solani was reduced significantly at of Academia Sinica 38, 33-39. 20 °C and 25 °C by the Trichoderma 4. Baker, R. (1989). Improved Tri-c isolate (Jn14). Similar results were obs- choderma spp. for promoting crop prod- served by Prasun and Kanthadai (1997) ductivity. Trends in Biotechnology 7, who reported that Trichoderma (isolate 34-38. Td-1) produced higher concentration of 5. Barakat, R. and Al-Masri, M. fungitoxic metabolites at higher temp- (2005). Biological control of gray mold peratures and effectively suppressed disease (Botrytis cinerea) on tomato the growth of S. rolfsii at or below 33 and bean plants by using local isolates °C. of Trichoderma harzianum. Dirasat, As a conclusion, this study showed that Agricultural Sciences 32(2), 145-156. some Trichoderma species and isolates 6. Barakat, R., Al-Mahareeq, F. and can be very efficient in controlling R. Al-Masri, M. (2006). Biological cont- solani damping off of various vegetab- trol of Sclerotium rolfsii by using in-d ble plants using several mode of actions digenous Trichoderma spp. isolates against the pathogen. Further studies from Palestine. Hebron University Re-s are needed on these promising fungi to search Journal 2(2), 27-47. identify potential compounds produced 7. Carling, D. E. (1996). Grouping and evaluate other possible mode of ac-t in Rhizoctonia solani by hyphal anas-t tions before going to field studies. tomosis interaction. Pages 35-46 in: Rhizoctonia Species: Taxonomy, Mo-l lecular biology, Ecology, Pathology and References: Disease Control. (B. Sneh, S. Jabaji- 1. Altomare, C., Norvell, W. A., Hare, S. Neate, and G. Dijst, eds). Kluw- Björkman, T., and Harman, G. C. wer Academic Publishers, Dordrecht, (1999). Solubilization of phosphates Netherlands. and micronutrients by the plant- growth 8. Chang Y-C, Chang Y-C, Baker, R., promoting and biocontrol fungus Tri-c Kleifeld, O., and Chet, I. (1986). In-c choderma harzianum Rifai 1295-22. creased growth of plants in presence of Applied Environmental and Microbiol-o biological control agent Trichoderma ogy 65, 2926-2933. harzianum. Plant Disease 70, 145-148. 2. Anusuya, D. and Jayarajan, R. 9. Chet, I. (1990). Biological Control (1998). Solubilization of phosphorus of Soilborne Pathogens with fungal by Trichoderma viride. Current Science antagonists in combination with soil treatment. In: Biological Control of Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 14

Soilborne Pathogens (pp. 15-25). D. The history and evolution of current Hornby, R. J. Cook, Y. Henis, WH. Ko, concepts. Plant Disease 87, 4-10. A. D. Rovira, B. Schippers, and P. R. 17. Inbar, J., Menende, A., and Chet, Scott (Eds.), New York: CAB Publishi- I. (1996). Hyphal interaction between ing House. Trichoderma harzianum and Sclerotin-i 10. Coley-Smith, J. R., Ridout, C. ia sclerotiorum and its role in biological J., Mitchell, C. M., and Lynch, J. M. control. Soil Biology and Biochemistry (1991). Control of button rot disease 29, 757-763. of lettuce (Rhizoctonia solani) using 18. Kleifeld, O., and Chet, I. (1992). preparations of Trichoderma viride, T. Trichoderma harzianum – interaction harzianum or tolclofos-methy. Plant with plants and effect on growth re-s Pathology 40, 359-366. sponse. Plant and Soil 144, 267-272. 11. Dennis, C., and Webster, J. 19. Laing, S. A. K., and Deacon, J. W. (1971a). Antagonistic properties of spec- (1991). Video microscopy comparison cies groups of Trichoderma. III. Hyphal of mycroparasitism by Pythium olig-a interaction. Transactions of British Myc- andrum, P. nunn and an un named Pyt- cological Society 57, 363-369. thium species. Mycological Research 12. Dennis, L., and Webster, J. 95, 469-479. (1971b). Antagonistic properties of 20. Mihuta-Grimm, L., and Rowe, R. species groups of Trichoderma. I. Prod- C. (1986). Trichoderma spp. As biocont- duction of non –volatile antibiotics. trol agents of Rhizoctonia damping-off Transaction of British Mycological Soc- of radish in organic soil and comparison ciety 57, 25-29. of four delivery systems. The American 13. Elad, Y., Chet, I., and Katan, J. Phytopathology Society 76, 306-312. (1980). Trichoderma harzianum: A 21. Noble, R. and Coventry, E. (2005). biocontrol agent effective against Scle-r Suppression of soilborne plant diseases rotium rolfsii and Rhizoctonia solani. with composts: A review. Biocontrol Journal of Phytopathology 70, 119- Science and Technology 15(1), 3-20. 121. 22. Papavizas, G. C. (1985). Trichode-r 14. Hadar, Y., Chet, I., and Henis, Y. rma and Gliocladium: biology, ecology, (1979). Biological control of Rhizocto-n and potential for biocontrol. Annual nia solani damping-off with wheat bran Review of Phytopathology 23, 23-54. culture of Trichoderma harzianum. 23. Prasun, K. and Kanthadai, R. Journal of Phytopathology 69, 64-68. (1997). Effect of temperature on an-t 15. Harman, G. E. (2000). Myths and tagonistic and biocontrol potential of dogmas of biocontrol: Changes in perc- Trichoderma sp. on Sclerotium rolfsii. ceptions derived from research on Tri-c Mycopathologia 139, 151-155. choderma harzianum T-22. Plant Dise- 24. Samuels, G. J. (1996). Trichoderm- ease 84, 377-393. ma: a review of biology and systematic 16. Howell, C. R. (2003). Mechanisms of the genus. Mycology Research 100, employed by Trichoderma species in 923-935. the biological control of plant diseases: 25. Shenker, M., oliver, I., Helman, Radwan Barakat et al., Bilogical Control.... H.U.R.J., Vol.(3), No.(1): 1-15 , 2007 15

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