African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 19, Num. 3, 2011, pp. 213-225

African Crop Science Journal, Vol. 19, No. 3, pp. 213-225

Potential Antagonistic Fungal Species From Ethiopia For Biological Control Of Chocolate Spot Disease Of Faba Bean

S. Sahile, P.K. Sakhuja¹, C. Fininsa¹ and S. Ahmed²

Department of Plant Science, University of Gondar P. O. Box 196, Gondar, Ethiopia ¹Department of Plant Science, Haramaya University P. O. Box 138, Dire Dawa, Ethiopia ²International Center of Agricultural Research in the Dry Areas, P. O. Box 5466, Aleppo, Syria
Corresponding author: hanasahile@yahoo.com

(Received 14 January, 2011; accepted 10 September, 2011)

Code Number: cs11021

ABSTRACT

Chocolate spot disease (Botrytis fabae Sard) is one of most yield limiting constraints of faba bean (Vicia faba). There is promise in using biological control agents to control chocolate spot diseases, nevertheless, this strategy has not been fully exploited. The objective of this study was to assess the prevalence of different antagonistic fungi on phyloplane of faba bean in Ethiopia and to evaluate their antagonistic potential against the pathogen. A total of 110 isolates of Trichoderma species were obtained from faba bean leaves from 12 districts, which were grouped into 18 distinct groups differing in colony and other characters. Similarly, 26 distinct isolates belonging to species of Penicillium, Aspergillus, Fusarium and Phioalophora were identified from leaves of faba bean. In vitro and in vivo studies revealed strong antagonistic potential of many isolates. Thirteen isolates of Trichoderma produced 4 mm or more inhibition zone and reduced growth of pathogen colony, when grown in dual culture with it. Antagonistic isolates caused lysis of pathogen mycelium more than 6 mm on agar plates. The antagonists significantly reduced pathogen growth in a range of 24.5 to 0.8 mm. The efficacy of the Trichoderma isolates ranged from 47.6 to 98% and that of the other fungal isolates ranged from 13.1 to 34.5%. On detached leaves, isolates 6-1T, 18-3T and 87T of T. ovalisporum and 52-BT, 108-1T and 108-4T of T. longibrachiatum were found to reduce development of chocolate spot on four genotypes of faba bean. The outcome indicates that biocontrol agents, particularly of species Trichoderma are prevalent on faba bean leaves and can be further explored and developed into effective mycofungicides for management of chocolate spot disease of faba bean.

Key Words: Aspergillus Botrytis fabae, Trichoderma, Vicia fabae

RÉSUMÉ

La maladie de tâche du chocolat (Botrytis fabae Sard) est une des contraintes limitatives du rendement du haricot faba (Vicia faba). L’utilisation des agents biologiques de contrôle serait promettant, par ailleurs, cette stratégie n’a jamais été amplement exploitée. L’objectif de cette étude était d’évaluer la prévalence de différents champignons anatagonistiques sur la phylloplane du haricot faba en Ethiopie et d’évaluer leur potentiel antagonistique contre le pathogène. Un total de 110 isolats d’espèces de Trichoderma était obtenu des feuilles du haricot faba dans 12 districts, et groupées en 18 groupes différents en colonie et autres caractères. Similairement, 26 différents isolats appartenant aux espèces de Penicillium, Aspergillus, Fusarium et Phioalophora étaient identifiées des feuilles de haricot faba. Des études in vitro et in vivo ont révélé un fort potentiel anatagonistique de beaucoup d’isolats.Treize isolats de Trichoderma ont produit 4 mm ou plus de zones d’inhiition et ont réduit la croissance de colonies pathogéniques lorsque cultivés ensemble. Sur plateaux agar, des isolats anatagonistiques ont causé plus de 6 mm de lysis des mycelium pathogéniques. Les anatagonistes ont significativement réduit de 24.5 à 0.8 mm la croissance des pathogènes. L’efficacité des isoltas de Trichoderma variait entre 47.6 et 98% et celle d’autres isolats fongiques variait entre 13.1 et 34.5%. Sur des feuilles détachées, les isolats 6-1T, 18-3T et 87T de T. ovalisporum et 52-BT, 108-1T et 108-4T de T. longibrachiatum étaient trouvées susceptibles de réduire le développement de la tâche du chocolat sur quatre génotypes du haricot faba. Le résultat indique que des agents biocontôles, particulièrement des espèces Trichoderma sont prévalants sur des feuilles de haricot faba et peuvent être examinés et développés en mycofungicides efficaces pour la gestion de la maladie de tâche du chocolat du haricot faba.

Mots Clés: Aspergillus Botrytis fabae, Trichoderma, Vicia fabae

INTRODUCTION

Faba bean (Vicia fabae L.) is one of the most important food legumes due to its high nutritive value both in terms of energy and protein contents (24-30 %) and is an excellent nitrogen fixer (Sahile et al., 2008a). Ethiopia is the third largest producers of faba bean in the world, next to china and Egypt (Torres et al., 2006). Faba bean is grown on 370,000 hectares in Ethiopia with an annual production of about 450,000 tonnes (ICARDA 2006). Despite its wide cultivation, the average yield of faba bean is quite low in Ethiopia, because of many biotic and abiotic constraints (Sahile et al., 2008a).

Chocolate spot is the most important disease of faba bean worldwide and is capable of devastating unprotected crops up to 67% (Bouhassan et al., 2004). It appears as reddish brown spots on leaves and under favorable conditions on stems, flowers and pods. Subsequently, these spots grow larger and can even merge into black mass. The disease results in heavy premature defoliation and under warm moist conditions crop lodging may occur. Plant growth and most physiological activities are adversely affected leading to drastic reduction in yield (Khaled et al., 1995). This disease is caused by Botrytis fabae Sard. and B. cinerea Pers.Fr. (Harrison 1988). In Ethiopia chocolate spot is caused by B. fabae and occurs almost regularly in most faba bean growing areas (Dangachew, 1967). The disease can reduce faba bean yields up to 67% (Bouhassan et al., 2004).

Different management options have been developed to reduce the yield losses in faba bean due to chocolate spot worldwide. These include the use of chemical fungicides, resistant/ tolerant varieties, use of certain cultural practices such as crop residue management and altering planting date (Dereje, 1999; Bretag and Raynes, 2004; Hawthorne, 2004). There is weak genetic resistance in cultivars of faba beans to chocolate spot (Lawes et al., 1983) and the most common control strategy is fungicide sprays. However, the negative effects of fungicide use are already becoming apparent. For instance, development of resistance in B. cinerea and in B. fabae against fungicides has been reported (Parry, 1990). Management options recommended in Ethiopia for this disease are application chlorothalonil or mancozeb and late planting (Dereje, 1993; Sahile et al., 2008b ) but have not been adopted by the farmers at large. Only one resistant variety, CS20DK was released in Ethiopia 20 years ago, but it did not become popular because of lower yield, and subsequently loss of resistance (Gemechu et al., 2006).

Biological control is another option, which has not been fully exploited. It is economical, self-perpetuating and usually free from residual effects and can be an important component of integrated disease management. Faba bean phyloplane harbours many microorganisms of different groups, besides the chocolate spot pathogen because of its high proteins content. Some of them might be antagonistic to B. fabae. Sherga (1997) found that out of 270 isolates of Bacillus tested, 14% had strong antagonistic effect against chocolate spot pathogen in vitro. However, fungal antagonists have not been explored for biological management of this serious disease of this important food legume.

The objective of this study was to identify the potential fungal antagonists to B. fabae from Ethiopia, which can be developed into commercial mycofungicides for the integrated management of chocolate spot disease of faba bean.

MATERIALS AND METHODS

Collection of leaf samples. Samples of healthy looking leaves were collected from faba bean plants having disease on other leaves, for exploring the resident antagonistic mycoflora of healthy faba bean leaves. Such samples were collected from farmers’ fields of twelve districts of Amhara Regional State in north western Ethiopia. This region is located between longitude 36 - 40^o W to E, and latitudes 11 - 13^o 45’ south-west to north. Similarly leaves aggressively affected by chocolate spot were collected from faba bean plants from a farmer’s field in Kutaber district for isolation of virulent pathogen. All the leaf samples were kept in folds of newspapers in a plant press for 48 hr and, thereafter, kept securely in labelled paper bags till isolation of the microorganisms.

Isolation of faba bean resident mycoflora and pathogen. The collected leaves of faba bean were surface sterilised in 1% sodium hypochlorite and subsequently washed three times in sterilised distilled water. From decontaminated leaves 5-mm² pieces were cut with sterile scalpel and placed on potato dextrose agar (PDA) in 9 cm diameter culture dishes. These were incubated for one week at 21^o C±1 and the fungi emerging from leaf tissues were transferred to PDA and purified. Fungal isolates in pure cultures were coded and transferred to screw-capped culture bottles containing faba bean extract dextrose agar and potato dextrose agar (FBEA/PDA) and stored at 4 °C. The B. fabae was isolated from diseased leaves on PDA, purified, identified and stored at 4 °C as stock culture of pathogen.

Cultural characteristics of isolates. The isolates of all fungi were grown on PDA in culture dishes at 21^o C ±1 for 96 hr. Characteristics like colony colour and diameter, morphological and sporulation of all the isolates were compared and recorded (Dhingra and Sinclair, 1986). Generic level identification of isolates was done and broadly classified into Trichoderma isolates and other fungal isolates. Different isolates within both groups showing similar colony characteristics were again grouped together and one representative isolate of each subgroup was taken for antagonistic potential studies. Cultural characteristics like growth rate, colony colour and diameter and medium reverse colour of all representative isolates of Trichoderma and other fungi were studied at 21^oC on PDA. Isolates of Trichoderma that showed promising antagonistic activity were identified from CAB International Global Plant Clinic, London, UK.

Antagonistic activity in fungal isolates against B. fabae. The antagonistic activity of different fungal isolates from apparently healthy leaves was tested against B. fabae firstly in vitro and then in vivo.

The fungi isolated from the leaves were tested for antibiosis activity to B. fabae on PDA in 9 cm Petri dishes. Three petri-dishes were inoculated with 2-mm mycelial disc from the edges of an actively growing colony of B. fabae on one side and with a similar sized disc of fungal isolate on the other side and incubated at 21^o C ±1. After 72 hr of growth the inhibition zones at the junction of colonies of fungal isolates and B. fabae were measured using verner calipper.

In order to test for lysis of B. fabae colony 4 ml of B. fabae mycelial disc was placed on PDA (15 ml) in 9 cm petri-dish and incubated at 21 ^oC ± 1. After three days of mycelial growth 2-mm agar disc of the potential fungal isolates of Trichoderma, and other fungal isolates from actively growing colonies were placed on the colony of B. fabae and incubated at 21^oC ± 1. Lysis of B. fabae colony was examined periodically under stereomicroscope (50x) and the width of lysed mycelia around the colony of the lytic fungal isolate was measured. The experiments were replicated three times in completely randomized design. Culture plates with B. fabae alone were used as the control.

Six Trichoderma isolates showing fast growth, significant inhibition zone and lytic activity were identified at CAB International Global Plant Clinic and used in the in vivo test of the antagonistic potential. Detached-leaves of four faba bean varieties namely CS20DK (tolerant), EH91011-6-2 (moderately resistant) and EH0013-18 (susceptible check) and one local check were used in vivo testing using the aggressive B. fabae culture according to the Paul et al. (1995) procedure. Leaves without antagonist evidence were used as the control.

Leaves of faba bean of the same age group were sterilised with 70% ethanol and placed in sterile 15 mm petri-dishes having sterilised filter paper moistened with sterile water. A spore suspension of B. fabae (2.5 x 10⁵ spores ml^-1) was prepared according to Mohammed et al. (1994). One drop (1 ml) of the spore suspension was placed near the midrib. The covered petri-dishes serving as moist chamber were incubated at 20^oC. After 24 hours, a drop of potential antagonist having 2.0 x 10⁵ cfu ml^-1 was added to the pathogen at the midrib and incubated at 20 ^oC again. The experiment was arranged in a complete randomised design.Disease reaction was recorded at 48, 72, 96 and120 hr of inoculation of antagonist using a 1-5 scale for detached leaf test (ICARDA, 1986).

Statistical analysis. Analysis of variance was carried out using SPSS V.12. Measurement data from in vivo test using detached leaves severity scales were subjected to SAS (Ver. 8). Mean comparisons were made using the Least Significant Difference test at P<0.05 test.

RESULTS

Isolation of resident mycoflora. From 1044 leaves collected from 12 districts, Trichoderma was isolated from 110 samples, showing 10.5% frequency of occurrence (Table 1). Trichoderma species was found to occur on faba bean leaves in all the districts; however, distribution of these isolates was found to vary among the districts. Highest number (13) of isolates was obtained from Kutaber district, followed by Hulet Eju Ensae. The elevation of the sampled areas varied between 1900 and 3319 meters above sea level.

Fungi other than Trichoderma species also appeared in isolations made from the 1044 leaves. Predominant fungi species observed on faba bean leaves were Penicillium, Aspergillus and Fusarium. Species of Penicillium were highly frequent and occurred on leaf samples from several areas. Aspergillus niger was isolated from 7 districts namely Debark, Desei Zuria, Kutaber, Farta, Wogera, Yilmana Densa and Hulet Eju Ensae while; A. flavus occurred in Kutaber, Lay Gaynt and Gonder Zuria districts. Similarly, Fusarium sp. occurred in Ambasel Tehuledrae, Gonder Zuria and Kutaber districts. Phailophora sp. was found to occur on faba bean leaves in Kutaber districts only (Table 1).

Cultural characteristics of isolates of Trichoderma species and other fungi.

Cultivation of fungal isolates on PDA under similar conditions showed that some isolates of the same genus resembled each other in colony characteristics, mycelium and sporulation characters while differed from other such groups of the same genus (Table 2). Eighteen distinct groups of Trichoderma were found to occur within its total 110 isolates from faba bean leaves. Further studies on PDA with one representative isolate of each group showed that colony diameters of 18 Trichoderma isolates varied from 39.4 to 61.55 mm after 96 hr of growth. Isolates 18-2T, 18-3T, 51b-T, 52-2T, 87T, 108-1T, 108-3T, 108-4T, 114-3T, 117-2T, 118T, 120-2T and 140-2T had fairly high growth rates. Isolates 51-bT, 1083T, 108-4T, 117-3T and 118T grew as suppressed colonies; while all others had raised aerial growth. Isolates 6-1T, 14-bT, 63T, 87T and 118T had white to green colour; while 51bT and 52-2T had grey to yellow colour (Table 2). All other isolates of Trichoderma were green in colour. There were distinct differences in media reverse colour of isolates, which varied from white, yellow, yellowish green to green. Species of Penicillium exhibited relatively slow growth rates with colony diameter ranging from 27.1- 31.2 mm in 96 hr of growth on PDA in comparison to A. niger and A. flavus with colony diameter of 32.2 - 48.4 and 33.2 – 37.2 mm, respectively. Phailophora sp. attained 41.3 mm colony diameter in the same period (Table 3).

Antibiosis activity in fungal isolates. Dual culture studies on PDA for evaluation of antibiosis activity of Trichoderma species and other fungi revealed that all inhibited growth of B. fabae by degrees and exhibited inhibition zone at the junction with the pathogen. Out of 18 Trichoderma isolates tested, 13 isolates viz., 61T, 14bT, 18-3T, 52-2T, 87T, 108-1T, 108-3T, 1084T, 117-2T, 118T, 120-2T, 122-1T and 140-2T produced 4 mm or higher inhibition zone (Table 4). Isolates of Trichoderma species reduced the growth of B. fabae colony by varying degrees. All Penicillium isolates produced 4-5 mm inhibition zones. Aspergillus niger isolates produced 5-6 mm inhibition zones in comparison to 4-5 mm by A. flavus and Fusarium species. Single isolate of Phailophora sp. produced 6 mm inhibition zone against B. fabae (Table 5).

Lytic potential in fungal isolates. All the Trichoderma isolates when placed on mycelium of B. fabae caused lysis to varying extent. Isolates 6-1T, 52-2T, 87T, 108-1T, 108-3T and 1202T caused 8-10 mm of lysis around them. Trichoderma isolates 6-1, 18-2, 18-3, 51-b, 52-2, 63, 87, 108-1, 108-3, 108-4, 114-3, 117-2, 117-3, 118, 120-2,122-1 and 140-2 overgrew upon the pathogen mycelium (Table 4).

Species of Penicillium placed on B. fabae also caused lysis of its mycelium, which ranged from 6.7 – 11.2 mm. Isolates 119-2, 62, 14, 29, 1342, 81, 10-p-2 and 119-B caused higher lysis ranging from 9-11.2 mm. Species of Aspergillus produced lysis ranging from 6-12.5 mm and isolates 49, 1403, 122-2, 56, 112, 30-1, 18-1 and 2 of A. niger and 68 of A. flavus showed higher lytic potential. Except for isolate 130-2, Fusarium species produced lesser lysis of mycelium of pathogen. Phailophora sp. proved effective in lysing the pathogen by 9.5 mm (Table 5).

Effect of Trichoderma species on chocolate spot in vivo. Effect of 3 isolates 6-1T, 18-3T and 87T belonging to T. ovalisporum and 3 isolates 52-BT, 108-1T and 108-4T of T. longibrachiatum on development of chocolate spot on four genotypes of faba bean was studied in vivo using detached leaf technique (Table 6). All isolates were found to reduce chocolate spot severity, when inoculated with the pathogen. However, their effect varied with isolate and genotype. Isolates 108-1T, 108-4T and 52-BT, of T. longibrachiatum were the most effective in reducing the mean disease severity on all the four genotypes and provided 43-47% mean disease control. Out of three isolates of T. ovalisporum, 6-1T proved better than other isolates of this species and reduced the disease by 40%, but was less effective than those of T. longibrachiatum. Isolates 18-3T and 87T of T. ovalisporum were very effective on susceptible genotypes EH91011-6-2, EH0013-18 and local check, but failed to reduce severity on moderately resistant genotype CS20DK. Highly significant disease control (75%) was provided by T. ovalisporum (isolates 108-1T and 108-4T) on genotype EH0013-18. Highest disease pressure developed on local check and all the 6 isolates could reduce disease severity. In general, all the isolates effectively reduced the disease on the two susceptible genotypes, but were less effective on moderately resistant genotypes EH91011-6-2 and CS20DK.

DISCUSSION

Trichoderma species predominantly occurred on faba bean leaves in Ethiopia. Species of Trichoderma were encountered from 10.5% leaf samples from 12 districts, indicating their natural adaptability to faba bean leaves. However, a variation in isolates of this genus was also found to be widespread. Within the total 110 isolates of Trichoderma species obtained from leaves, 18 distinct isolates showing clear differences in colonies and morphology were established. Besides, Trichoderma, 26 distinct isolates of Penicillium, Aspergillus, Fusarium and Phailophora were also found to be prevalent on faba bean leaves. A total of 11 isolates belonging to A. niger and A. flavus, 10 isolates of Penicillium, 4 isolates of Fusarium and one isolate of Phialophora were obtained from faba bean leaves. There was no co-relation of species of fungi with altitude as all of them occurred at all altitudes from where samples were collected. This clearly indicated their wide adaptability to different environments. Goldfarb et al. (1989) reported the varying nature of the growth rate of Trichoderma with species and temprature. In their study, Goldfarb and his co-workers found the growth rate of Trichoderma spp. to vary from 12.7-23.4 mm day^-1 depending on the species at 20 ⁰C of temprature. In another experiment, Saber et al., (2009), found daily growth rate of different fungal antagonist in the range of 15-35 mm/day. In their experiment conducted in Egypt, the author reported that all of the fungal antagonists tested showed reasonably higher growth rate than the pathogen B. fabae.

The dual culturing of pathogen with 18 isolates of Trichoderma and 26 of other fungi revealed clearly potential of control in some of the isolates. Thirteen isolates of Trichoderma produced 4 mm or higher inhibition zone on agar medium. These might be producing antibiotics or extracellular enzymes, which inhibited growth of the pathogen. Similar strong antagonistic behaviour of some isolates was observed in lysing the pathogen mycelium in agar plates. Some isolates proved effective in antibiosis as well as in lysis, while some others were better in antibiosis and some better in lysing. This reflects the differences in the spectrum and degree of their antibiotic and enzyme production. The genus Trichoderma comprises a great number of fungal strains that act as biological control agents, the antagonistic properties of which are based on the activation of multiple mechanisms. Elad and Stewart (2004) have also reported that Trichoderma, Gliocladium and Ulocladium have greatest potential for Botrytis diseases and commercial success has been achieved in glasshouse and post-harvest environments for disease control. In the activity of biological control, micro-organisms action is not limited to direct influence on the target diseases, in addition to their direct effect they also enhance the resistance of the plants. A report by Benítez et al. (2004) indicates that Trichoderma strains are known to promote plant growth and plant defensive mechanisms and antibiosis against, the pathogen or direct mechanisms such as mycoparasitism. T. harzianum and T. viridi have been reported as biocontrol agents for chocolate spot of grape, apple and strawberry caused by B. cinerea (Sutton et al., 1997; Hjeljord et al., 2001).

The study showed that there were promising antagonistic species of fungi prevalent on faba bean leaves, which can be exploited for the control of chocolate spot. Although, different genera of fungi were found to have antagonistic ability against the pathogen in vitro, from the point of view of wider antagonistic spectrum, Trichoderma species were considered more feasible for further exploration. Therefore, isolates 6-1T, 18-3T and 87T of T. ovalisporum and 52-BT, 108-1T and 108-4T of T. longibrachiatum were further tested in vivo by detached leaf technique. All of them were found to reduce the development of chocolate spot on two susceptible and two moderately resistant genotypes of faba bean, though degree of reduction varied and also depended on genotype of faba bean. Isolates provided a higher extent of control in susceptible genotypes. These antagonistic isolates were fast growing and reduced the colony growth of B. fabae, when grown in dual culture.

This study has revealed isolates of T. ovalisporum and T. longibrachiatum as effective antagonists of B. fabae for the first time. Trichoderma ovalisporum is an endophytic type of fungus and was first identified as a new and novel biocontrol agent from Amazon basin of South America for frosty pod rot (Moniliopthora rori) and wittches’ broom (Crinipellis spp.) of cocoa (Holmes et al., 2004; Holmes et al., 2006).

Trichoderma strains are known to control pathogens either indirectly by competing for nutrients and space, modifying the environmental conditions, or promoting plant growth and plant defensive mechanisms and antibiosis, or directly by mechanisms such as mycoparasitism (Benítez et al., 2004). Trichoderma species such as T. harzianum, T. viridi and T. polysporum are well known antagonists. Ten commercial products of these three species were developed for controlling diseases on different crops (Frevel et al., 1998). T. harzianum and T. viridi were reported as biocontrol agent for chocolate spot of grape, apple and strawberry caused by B. cinerea (Sutton et al., 1997; Hjeljord et al., 2001).

Other fungi prevalent on faba bean leaves also exhibited antagonistic activities against B. fabae in vitro. Isolates 62, 29, 10-p2 of Penicillium; 140-3, 122-2, 56, 18-1 and 112 of A. niger, 25 of A. flavus and 24 of Fusarium and 120-2yl of Phailophora caused wide inhibition zone and lysis of mycelium. The antagonists evaluated in this study showed significant differences in reducing pathogen growth and their effects ranged from 24.47 to 0.8 mm (Table 5). Earlier also Penicillium brevicompactum and Cladosporium cladosporioides isolated from faba bean leaves were found to have significant antagonistic activity against B. fabae in vitro and in vivo (Jackson et al., 1997). Commercial products like Biofox C and Fusaclean having nonpathogenic strains of Fusarium oxysporum have been developed for controlling soil borne diseases (Frevel et al., 1998). De Cal et al. (2008) reported biological control of powdery mildew on strawberry leaves by Penicillium oxalicum applications, it was achieved on different cultivars and lines in growth chambers and in open-field nurseries. Species of Penicillium, Aspargillus and Fusarium have been reported by Leibinger et al. (1997) as an antagonists against Botrytis cinerea. Mass production technology by solid state fermentation for conidi of Penicillium frequentans, a biocontrol agent of the fungal pathogen Monilinia laxa has been developed by using specially designed plastic bags (VALMIC ® ) containing peat and vermiculite (De Cal et al., 2002 ).

ACKNOWLEDGMENT

This research was financed by IFAD and Haramaya University SIDA-SAREC support, Ethiopia. The authors thank Haymanot Bezuneh, Haramaya University, Yenework G/medhin Adet Agricultural Research Center, for their assistance in data collection and laboratory works.

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