Management of Castor Gray Mold Using Trichoderma Species

Castor gray mold caused by Botryotinia ricini is one of the most destructive diseases of castor causing heavy yield losses. Ecologically sustainable approaches towards tackling diseases are gaining importance owing to ill-effects of chemical pesticides. One among them is biological control through application of fungal and bacterial antagonists that has wide range applicability against plant diseases. Biological control using antagonistic microorganisms is a potential ecofriendly and sustainable approach, and the effective bioagents can be applied for the efficient management of castor gray mold to ensure better returns. Keeping in view the importance of disease in castor production, the present investigation was carried out to identify the Trichoderma species against B. ricini under in vitro and glass house conditions. The antagonistic activity of twelve Trichoderma isolates was evaluated against Amphobotrys ricini under laboratory and greenhouse conditions. Among the Trichoderma isolates tested, Trichoderma asperellum isolate 1, T. harzianum isolate 5 and T. asperellum isolate 3 recorded significantly highest per cent inhibition of mycelial growth of A. ricini. These Trichoderma isolates were found to have high chitinase and glucanase activity compared to other isolates tested. Further, greenhouse experiments have revealed the efficacy of T. harzianum isolate 1, T. asperellum 2, T. harzianum isolate 4 and T. harzianum isolate 3 in reducing gray mold severity. The antagonistic isolates were found to have high chitinase and glucanase activity.


INTRODUCTION
Castor (Ricinus communis L.), is an important non-edible oilseed crop. It contains 50-55% oil and plays a vital role in Indian vegetable oil economy. It is mainly grown in tropical and sub-tropical climate. Castor is known to suffer from many fungal and bacterial diseases at different crop growth stages. Among these, one of the most destructive diseases of castor, primarily infecting inflorescence and racemes, is gray mold, caused by the fungus, Amphobotrys ricini (N.F. Buchw.) Hennebert. Yield losses are extensive due to this disease and are a major threat to commercial cultivation of the crop. Seed yield loss up to 100% was reported from India (Soares, 2012).
Plants are surrounded by a diverse range of organisms in their environment, including bacteria, fungi, oomycetes, nematodes, insects and viruses. Although some of these organisms may have a negative impact on the plant, others may exert beneficial effects by enhancing the general fitness of the plant and/or by suppressing plant disease. Research on such biocontrol organisms has intensified during recent decades and their importance has increased as a part of integrated management practices to reduce chemical pesticide use (Glare et al., 2012).
Over the past century, growers have relied heavily on the use of chemicals to control diseases caused by Amphobotrys species. Fungicide resistance coupled with current public concern for both the environment & pesticide residues in food has highlighted the need for alternative methods for disease control and biological control is one such approach. Biological control may result from direct or indirect interactions between the beneficial microorganisms and the pathogen (Benítez et al., 2004;Viterbo et al., 2007). A direct interaction may involve physical contact and synthesis of hydrolytic enzymes, toxic compounds or antibiotics as well as competition. An indirect interaction may result from induced resistance in the host plant, the use of organic soil amendments to improve the activity of antagonists against the pathogens (Viterbo et al., 2007).
The genus Trichoderma comprises a great number of fungal strains with biocontrol capacity. They have adapted to diverse environmental conditions and are often the most frequently isolated fungi from soil (Harman et al., 2004). They are prolific producers of extracellular proteins, including enzymes that degrade cellulose and chitin (Nagy et al., 2007). Moreover, their capacity to reduce plant disease has been studied intensively, with both direct and indirect effects on plant pathogens (Lorito et al., 2010). Their high reproductive capacity, ability to survive under unfavourable conditions and high nutrient utilization efficiency contribute to their success as biocontrol agents (Benitez et al., 2004).
There are several reports on the use of Trichoderma species as biological agents against plant pathogens (Harman et al., 2004). Trichoderma species (T. harzianum, T. viride, T. virens etc.) have been used as biological control agents against a wide range of pathogenic fungi like Rhizoctonia, Pythium, Botrytis, Fusarium, Phytophthora, etc. (Benítez et al., 2004;Zeilinger & Omann, 2007). T. harzianum Th4d and T. asperellum Tv5 were most effective in inhibiting the pathogen. The growth inhibition was attributed to the production of mycolytic and defense related enzymes by the Trichoderma species (Navaneetha et al., 2015). The present investigation was undertaken to evaluate the effect of Trichoderma species against A. ricini under laboratory and greenhouse conditions.  (Mortan and Sproube, 1955). Per cent inhibition of mycelial growth of A. ricini was calculated as per the formula of Vincent (1927).

Determination of chitinase and glucanase activity of potential antagonists
Plate assay for determining the chitinase activity was conducted using 500 ml mineral salt solution, 500 ml of distilled water, 0.02% yeast extract, 15 g of agar and 2.4% of purified chitin (N-acetyl glucosamine from crab shells, Sigma chemical Co., St. Louis) as described by Campbell and Williams (1951). The assay for determining the activity of glucanase was conducted using the same medium as described for chitinase activity, but substituting the chitin with 0.5% (w/v) laminarin. Assay procedure All plates containing chitin and glucan were inoculated with the antagonists and were incubated at room temperature for 4-6 days and then flooded with 1% congo red solution in water. The stain was removed after 30 min and plates were destained with 1M Nacl in buffer solution for 15 min (Hagerman & Butler, 1985). Clear zones developed in the opaque agar around the colonies indicated the degradation of the substrate. The isolates were classified into different groups based on the zone of clearance (Low chitinase/ glucanase activity -<0.5 cm, medium chitinase/glucanase activity -0.51 to 0.99 cm and high chitinase/glucanase activity->1.0 cm). Plates which were not flooded with any of the stains described above, served as control.

Evaluation of Trichoderma spp. against A. ricini under green house conditions
The Trichoderma spp. were screened for their biocontrol potential against A. ricini using detached spike assay in a closed polythene humid chamber. Racemes/ spikes were collected from castor plants (var. DCH-519) and were kept in conical flasks containing 2% sucrose solution and are arranged on a green house bench in a randomized complete block design (RCBD) and replicated thrice. Racemes were sprayed with conidial suspension (10 7 conidia/ml) of Trichoderma spp. and inoculum (10 6 conidia/ml) of A. ricni was sprayed 24 hours after pathogen inoculation. Racemes sprayed with water alone or inoculated with spore suspension (10 6 conidia/ml) of A. ricini alone served as healthy and inoculated controls. Treated racemes were transferred to polythene humid chamber with fogging devices in which temperature and humidity were maintained at 22+2 0 C and relative humidity at 90%. Per cent infected capsules in each treatment were calculated when maximum number of capsules was infected in control using the formula: Per cent infected capsules = Number of infected capsules in a raceme X 100 Total number of capsules in a raceme The disease progress in various treatments was assessed using the disease severity scale given by Mayee and Datar et al. (1996) when maximum disease severity is observed in inoculated control.

Evaluation of Trichoderma spp. against A. ricini under in vitro conditions
Five isolates of Trichoderma asperellum and seven isolates of T. harzianum obtained from the Department of Plant Pathology, College of Agriculture, Rajendranagar were screened for their antagonistic potential against A. ricni by dual culture method. The perusal of the data in Table 1 indicated that all the Trichoderma isolates tested were highly effective in inhibiting the mycelial growth of A. ricini. Among the Trichoderma isolates tested, Trichoderma asperellum isolate 1, T. harzianum isolate 5 and T. asperellum isolate 3 (77.96, 77.41 and 77.04 per cent, respectively) recorded maximum inhibition of mycelial growth of A. ricini which were found statistically on par with each other. These were followed by T. harzianum isolate 4 with mycelial growth inhibition of 75.56 per cent.
Trichoderma harzianum isolate 2, T. asperellum 2 and T. asperellum 4 were statistically on par with each other and recorded an inhibition of mycelial growth by 68.15, 67.60 and 65.92 per cent, respectively. These were followed by T. harzianum isolate 3, T. harzianum isolate 7 and T. asperellum isolate 5 with mycelial growth inhibition of 65.00, 62.59 and 62.22 per cent. Least inhibition of mycelial growth of test fungus was observed with T. harzianum isolate 6 (56.85%) and T. harzianum isolate 1 (58.52%) which were statistically on par with each other The present investigations are in agreement with the findings of Raoof et al. (2003) who tested the efficacy of different strains of Trichoderma spp. against Botrytis ricini under in vitro conditions. Highest inhibition of the test pathogen was observed in T. asperellum treatment followed by T. koningii under dual culture assays and the inhibition of pathogen by Trichoderma spp. was observed 72 hours after incubation and after 96 hours, the hyphae of Trichoderma overgrew on B. ricini. Similarly, the effectiveness of T. viride and T. harzianum was established by Bhattiprolu and Bhattiprolu (2006). Navaneetha et al. (2015) conducted extensive studies on the efficacy of Trichoderma species against A. ricini and found that T. harzianum Th4 and T. asperellum Tv5 were most effective in inhibiting the pathogen. The growth inhibition was attributed to the production of mycolytic enzymes by the Trichoderma species.

Screening of Trichoderma isolates for chitinase production on solid medium supplemented with colloidal chitin
Chitinase activity exhibited by 12 Trichoderma isolates was determined by the diameter of the clear zone developed around the colonies indicating the degradation of colloidal chitin to N-acetyl glucosamine and the results are presented in Table 2. The Trichoderma isolates, T. asperellum isolate 1, T. asperellum isolate 3 and T. harzianum isolate 5 represented high chitinase activity group with 1.43, 1.23 and 1.63 cm zone of clearance on medium amended with colloidal chitin. Four isolates (T. asperellum isolate 4, T. asperellum isolate 5, T. harzianum isolate 4, T. harzianum isolate 6, T. harzianum isolate 7) expressed medium chitinase activity. Low chitinase activity was observed in the isolates, T. asperellum isolate 2, T. harzianum isolate 1, T. harzianum isolate 2, T. harzianum isolate 3, T. harzianum isolate 6 and T. harzianum isolate 7.
Trichoderma species are known to produce chitinolytic enzymes and are most effective agents of biological control of plant diseases (Agarwal & Kotasthane, 2012). In the present investigation, high chitinolytic strains of Trichoderma, T. asperellum isolate 1, T. asperellum isolate 3 and T. harzianum isolate 5 have shown maximum inhibition of mycelial growth of A. ricini compared to medium or low chitinolytic isolates under in vitro conditions.

Screening of Trichoderma isolates for glucanase production on solid medium supplemented with laminarin
Glucanase activity was determined for twelve isolates of Trichoderma and it was observed that a clear zone developed around the colonies on medium amended with laminarin. The data in Table 3 showed that T. asperellum isolate 1 produced maxuimum β-1,3-glucanase activity with 1.18 cm zone diameter followed by T. asperellum isolate 3 (0.95 cm) and T. harzianum isolate 5 (0.83 cm). Among the test isolates, the least β-1,3-glucanase activity was recorded in T. harzianum isolate 1 (0.38 cm).
The extracellular enzymes produced by Trichoderma isolates can be correlated with the antagonism. Elad et al. (1982) reported that the isolates of T. harzianum differ in their ability to suppress plant pathogens based on the levels of mycolytic enzymes produced by them. Results from the present study revealed that most of the isolates have shown good enzymatic activities. Trichoderma directly attacks the plant pathogen by excreting lytic enzymes such as chitinases, β-1,3-glucanases, proteases and also with the production of volatile and non-volatile metabolic compounds.

Effect of Trichoderma spp. on castor gray mold severity under green house conditions
Five T. asperellum isolates and seven T. harzianum isolates were evaluated under green house conditions for their efficacy against gray mold of castor using detached spike technique. The results are presented in Table 4. The perusal of the data indicated that all the treatments have significantly reduced the per cent infected capsules compared to inoculated control except T. harzianum isolate 2 and T. harzianum isolate 5. The per cent infected capsules were minimum in racemes preinoculated with Trichoderma isolates (Plate 1), T. harzianum isolate 1 (5%), T. asperellum 2 (6.67%), T. harzianum isolate 4 (6.67%) and T. harzianum isolate 3 (12%) which were statistically on par with each other. These were followed by T. asperellum isolate 4 (16.33%) which is significantly at par with T. asperellum isolate 5 (20.67%). There was no significant difference between the raceme sprays with T10 (25.0%) and T. harzianum isolate 7 (29.00%). More than fourty per cent of the capsules were infected in racemes sprayed with T. asperellum isolate 3 (41.67%) and T. asperellum isolate 1 (50.0%). The per cent infected capsules were maximum in racemes sprayed with T. harzianum isolate 2 (86.0%) and T. harzianum isolate 5 (91.33%), which were on par with inoculated control. The efficacy of various treatments in terms of disease control is depicted in Fig.1.
The results are in accordance with Raoof et al. (2003) who tested the efficacy of different strains of Trichoderma spp. against Botrytis ricini under green house conditions using detached spike method. It was observed that spraying of T. asperellum on castor racemes reduced the disease up to 45 per cent. Similar results were also obtained by Bhattiprolu and Bhattiprolu (2006) under field conditions by spraying castor spikes with T. viride (10 6 spores ml -1 ) immediately after the disease appearance. Antagonism of three Trichoderma species (T. harzianum, T. viride and T. longibrachiatum) was evaluated against Botrytis fabae and B. cinerea, the causal agents of chocolate spot of Faba bean (Vicia faba L.) in Algeria. Among the Trichoderma isolates tested, T. harzianum and T. longibrachiatum were shown to over grow the colony of Botrytis, whereas T. viride was known to suppress the Botrytis species by antibiosis (Bendahmane et al., 2012). Research on such biocontrol organisms has intensified during recent decades and their importance has increased as a part of integrated management practices to reduce chemical pesticide use (Glare et al., 2012).