Evaluation of three species of trichoderma as potential bio-control agent against colletotrichum gloeosrioides., a casual agent of anthracnose disease in onion.
The research was conducted to screen and evaluate the effect of three Trichoderma species as a potential bio-control agent against Colletotrichum gloeosrioides isolated from onion under in vitro condition. In in vitro test, antagonistic effect of the three Trichoderma species against C. gloeorioides was evaluated using dual culture test and the presence of active compounds of the three antagonists was determine while mycoparasitism interaction was also observed under microscope using slide culture technique. Pathogenicity testing was also determined using onion bulb as test crop. Results showed in the percent growth inhibition of the three Trichoderma species during 14 days of incubation, T. longibrachiatum obtained the highest percentage inhibition of 64.68% compared to T. harzianum and T. asperellum with 59.16% and 47.73% inhibition respectively. It was also observed that hypal coiling was common on the three Trichoderma species against C. gloeosrioides. The presence of active compounds among the three Trichoderma were also tested and it showed at 500 [micro]l/ml crude extract of T. longibrachiatum obtained the highest inhibition of 15.58% which make it more active against a wide range of microorganism. Pathogenicity test of C. gloeosrioides against the three Trichoderma showed the onion bulbs inoculated with the pathogen only was highly pathogenic. However, bulbs treated with the antagonist showed that C. gloeosporioides was observed as weak and mild pathogenic. This could be concluded that the presence of Trichoderma species could lessen the pathogenic effect of C. gloeosporioides and could be used as natural biocontrol agent of some plant diseases.
KEYWORDS: Trichoderma, Bio control, Anthracnose, Onion
Onion (Allium cepa L.) under the Liliaceae family is either a biannual or perennial plant, bearing semi-cylindrical leaves which emerged from a subterranean bulb and having fascicled, short, branched roots. During the cultivation process of onion in the farm, onions are very susceptible to pathogens especially when the environmental conditions favored their growth. Once infected by fungi or bacteria, it can lead to reduce quality and productivity of onion . In the past year, Colletotrichum species, the causal agent of anthracnose seriously damaged several onion fields in Nueva Ecija as well as neighbouring provinces. Anthracnose was responsible for severe leaf blighting in some fields . This resulted to yield loss, high price and shortage of onion supply in the market.
Biological control is a component of an integrated pest management strategy. It is defined as the reduction of pest populations by natural enemies and typically involves an active human role. This is referred to as natural control or biological control which includes the use of predators, parasitoids, and pathogens. Biological control agents of plant diseases are most often referred to as antagonists .
The genus Trichoderma is very popular in organic farming. It is employed widely in agriculture because it releases a variety of compounds that induce systematic resistance against soil- borne pathogen, and enhances crop productivity .
Thus, this study is designed to determine if the three species of Trichoderma can be a potential bio-control agent against C. gloeosporioides. in onion. Further, the effect of these natural extracts in onion subjected to C. gloeosporioides was also determined.
MATERIALS AND METHODS
Collection and isolation of C. gloeosporioides causing disease in onion:
Prior to isolation, advancing disease portion was cut into 1 cm[.sub.2] section, disinfected and washed with sterile water for 3 minutes. The tissues were blot dried on sterile filter paper, then plated on water agar and incubated for 7 days at room temperature.
Morphological and cultural characterization of pathogen:
The culture of the pathogen was identified morphologically and culturally. The colony of the culture of the pathogen grown in previously plated Potato Dextrose Agar (PDA) was described and also the spore was observed under the compound microscope. Then, it was submitted to the Laboratory Service Division, Philippine Center for Postharvest Development and Mechanization, Science City of Munoz, Nueva Ecija for verification of the identification made.
Different species of Trichoderma such as T. longibrachiatum, T. asperellum and T. harzianum were used as antagonist against fungal pathogen causing disease in onion. Pure cultures of the three species of Trichoderma were obtained from the microbiology laboratory of RM-CARES.
Evaluation of the antagonists against Colletotrichum sp. in onion:
The antagonistic effect of the test antagonist on the growth of fungal pathogen causing diseases in onion was evaluated using dual culture method. Five mm mycelial plugs of each antagonist and fungal pathogen was aseptically inoculated equidistantly in previously plated petri dishes containing PDA. Plates inoculated only with the test pathogen served as control. Four plates for each interaction were incubated at room temperature. The percent growth inhibition (GI) was computed after 14 days of incubation as follows based on the study of Korsten et al.  as cited by Alvindia and Natsuaki (2008):
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Kr- distance (measured in mm) of fungal growth from the point of inoculation to the colony margin of the control plates;
rl- distance of fungal growth margin in the direction of the antagonist
Mycoparasitism interaction of each antagonist against fungal pathogen was determined using slide culture techniques. Agar blocks of the antagonists and fungal pathogens were grown equidistantly with a distance of 1 cm in a glass slide containing a very thin water agar. The slides were placed in sterile petri dish line with moist sterile filter paper. Slides were observed under the compound microscope after two weeks to determine the interaction of the antagonists and pathogens.
Test for the active compounds of the antagonists against fungal pathogen:
The active compounds of the antagonists were determined following the method of Talubnak and Soytong, . The antagonist was cultured in potato dextrose broth (PDB) for 14 days. Biomass of the antagonist was filtered and collected for extraction method using rotary vacuum evaporator. One ml of different concentration of crude extracts (0, 100, 500 and 1,000 [micro]l/ml) of antagonist were tested in petri dishes. Four mm mycelial disk of the pathogen was inoculated in the center of petri dish containing PDA and different concentration of crude extracts of microbial antagonist and incubated at room temperature. Colony diameter was recorded daily. Growth inhibition was also recorded and computed using the formula:
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
Kr- Distance (measured in mm) of fungal growth from the point of inoculation to the colony margin of the control plates.
rl- Distance of fungal growth margin in the direction of the antagonist; and
Gl- Percent inhibition growth
All the data collected were analyzed with Analyses of Variance (ANOVA) using Completely Randomized Design (CRD) with four replications. Duncan's Multiple Range Test (DMRT) was used in comparing means at 5% level of significance.
Pathogenicity test (onion bulb):
Four onion bulbs were washed with sterile water and disinfected with 10% sodium hypochlorite solution for 3 minutes and then rinsed again with sterile water and air dried. Four mm of 7 day old fungal pathogen and antagonist were attached side by side to the surface of the onion bulb. The control was inoculated only with agar disk. The bulb was kept inside the moist chamber for 4-5 days; the diameter of the lesions was recorded at 5th day of incubation. Disease severity of the onion bulb was determined using the grading scale of Villa Nova et al. .
RESULTS AND DISCUSSION
Morphological and cultural characteristics of the pathogen:
The color of C. gloeosporioides varied from white to grey. The growth pattern was either circular with the mycelia showing a uniform growth pattern and radial in a ring like pattern. The cultures on PDA grew well with growth rate of 80.93 mm after 7 days of incubation. The mycelium was hyaline, brown or both, sometimes abundant, at times sparse with floccose, loose or compact growth. In this study, based on cultural and morphological identification the isolate was identified as C. gloeosporioides.
The result of the dual culture of antagonist and pathogen showed that the three species of Trichoderma namely T. longibrachiatum, T. harzianum and T. asperellum had antagonistic effect against C. gloeosporioides (Table 1).
The bio control activity by Trichoderma can occur by means of several antagonistic mechanisms such as nutrient competition, antibiotic production, and mycoparasitism [8, 9].
Mycoparasitism has been proposed as the major antagonistic mechanism displayed by Trichoderma species .
Based on the dual culture test, all of Trichoderma isolates inhibited the mycelia growth of C. gloeosporioides with varying efficiencies (Table 2). T. longibrachiatum obtained the highest percent growth inhibition after 14 days of incubation with a mean value of 64.68% while the lowest growth inhibition was rated in T. asperellum.
A microbial biological control agent may express different mechanisms against pathogen during their antagonistic activity. It weakens or destroys the pathogen, parasitize the pathogen directly, produce antimicrobial compounds, compete for space and nutrients and produce enzymes that attack the cell components of the pathogen . Antibiosis, production of antibiotic compounds and inhibition of other microbes, is the most important mechanism expressed by the antagonistic bacteria . In this study, the antagonistic activity expressed by Trichoderma species in dual culture method might be due to the one or combination of all above mechanisms.
Figure 2 shows the interaction exhibited by Trichoderma species against the pathogen C. gloeosporioides. Hyphal coiling of three Trichoderma species on the hyphae of C. gloeosporioides was observed.
The Trichoderma species are useful avirulent plant symbiots that act as bio control agent against phytopathogenic fungi via mechanisms of competition, rhizophere competence, mycoparasitism, antibiotic and enzyme production, induced resistance, and promoting plant growth [13, 14,15]. The majority of Trichoderma species are antagonist of phytopathogenic fungi and have been broadly used as the most important bio control agent .
[FIGURE 2 OMITTED]
Mycoparasitism involves morphological changes, such as coiling and formation of appressorium-like structures, which serve to penetrate the host . After host recognition, Trichoderma species attaches to the host hyphae via coiling and penetrate the cell wall by secreting cell wall-degrading enzymes .
Test for active compounds:
The percent inhibition obtained by the different concentrations of crude extracts from Trichoderma was evaluated as shown in Table 3. T. asperellum at 500 [micro]l/ml crude extract concentration (TA 500) significantly obtained the highest percentage with a mean value of 24.40%. on the other hand, the lowest growth exhibited was noted in TA 1000 with a mean of 8.62%. Statistical analysis recorded significant differences among treatments evaluated.
Increasing the concentration of T. harzianum (TH1000) to 1000 [micro]l/ml showed no significant differences with TH 100 with the recorded inhibition rate of 11.53% and 11.22%, respectively. However, the result was significantly comparable with the result obtained from T. harzianum at a lower concentration of 100 [micro]l/ml with 11.22% inhibition rate.
Trichoderma species produce various volatile and non-volatile antimicrobial compounds which are active against a wide range of microorganisms . Growth inhibition of the pathogens by the Trichoderma metabolites has been reported by several researchers [14, 18].
Pathogenicity test (onion bulb):
Pathogenicity test was performed to determine the ability of Trichoderma to inhibit the growth of C. gloeosporioides in vivo. Table 4 shows the disease severity of the onion bulb.
The inoculated onion bulb without antagonist recorded the highest lesion with a mean of 26.27 mm and highest disease rating of 4 which is highly pathogenic. On the other hand, onion bulb exhibited the smallest lesion with a mean of 3.89 mm and disease rating of 1.
Trichoderma grows fast and compete with disease causing fungi for food, space as well as developing mycotoxin system for many soil or foliar pathogen , as well as stimulatory effect on growth of onion seedlings, seed germination, vigour index and fresh weight of seedlings . It is well documented that the interaction of Trichoderma species with the plant may promote growth, improves crop yield, increase nutrient availability and enhance disease resistance [21,22]. In addition, some species of Trichoderma can colonize root surfaces, interact with the plant and exchange compounds that can cause substantial changes in plant metabolism . Gajera et al.  reported that Trichoderma are develop exactly on other fungi's hyphae, coils around them and degrades the cell's walls. The action of parasitism restricts the development and activity of pathogenic fungi as well as produce secondary metabolites like cellular exochitinases [25,26] with antibiotic activity [13,27,28].
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(1) Hanna Jay A. Galindez, (3) Lani Lou Mar A. Lopez, (2) Soforonio P. Kalaw, (2) Kristine Grace D. Waing, (3) Jonathan L. Galindez
(1) Former Undergraduate Student, Department of Biological Sciences, College of Arts and Sciences,Central Luzon State University,Science City of Munoz, Nueva Ecija Philippines
(2) Faculty, Department of Biological Sciences, College of Arts and Sciences, Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines.
(3) Faculty, Ramon Magsaysay-Center for Agricultural Resources and Environment Studies (RM-CARES), Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines
Address For Correspondence:
Lani Lou Mar A. Lopez, Faculty, Ramon Magsaysay-Center for Agricultural Resources and Environment Studies (RM-CARES), Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines
Table 1: Types of interaction of the antagonists and pathogen Non-pathogenic Fungi Pathogenic Fungi Grade 1. T. longibrachiatum (+) C. gloeosporioides (-) Grade 2 2. T. harzianum(+) C. gloeosporioides (-) Grade 2 3. T. asperellum(+) C. gloeosporioides (-) Grade 2 Non-pathogenic Fungi Interaction 1. T. longibrachiatum (+) Mutual intermingling 2. T. harzianum(+) Mutual intermingling 3. T. asperellum(+) Mutual intermingling Note: (+)= aggressor (-)= victim; Grade 1= Mutual intermingling without any microscopic sights of interaction; Grade 2= Mutual intermingling growth where the growth of the fungus is ceased and bring over growth by the opposed fungus; Grade 3= Intermingling growth where the fungus under observation is growing into the opposed fungus either above or below; Grade 4= Sight inhibition of both the interacting fungus with narrow demarcation line (1-2); Grade 5= Mutual inhibition of growth at a distance of >2 mm Table 2: Percent growth inhibition of the three Trichoderma species against C. gloeosporioides Antagonist Percent inhibition after 14 days of inoculation (%) T. longibrachiatum 64.68 (a) T. harzianum 59.16 (b) T. asperellum 47.73 (c) (*) Means with different superscript are significantly different at 5% level of significance using DMRT Table 3: Percent growth inhibition of crude extracts of Trichoderma species Crude extract concentration ([micro]l/ml) Growth inhibition (%) TA100 9.23 (e) TA500 24.40 (a) TA1000 8.69 (e) TL100 9.00 (e) TL500 14.49 (c) TL1000 12.00 (d) TH100 11.22 (d) TH500 15.58 (b) TH1000 11.53 (d) (*) Means with different subscript are significantly different at 5% level of significance Table 4: Disease severity of the onion bulb Treatments Mean of Lesions Grading Control (uninoculated and untreated) (mm) Scale C. gloeosporioides 0 0 T. longibrachiatum vs. C. gloeosporioides 26.27 4 T. harzianum vs. C. gloeosporioides 5.04 2 T. asperellum vs. C. gloeosporioides 3.89 1 4.05 1 Treatments Indication Control (uninoculated and untreated) Non- pathogenic C. gloeosporioides Highly pathogenic T. longibrachiatum vs. C. gloeosporioides Mildly pathogenic T. harzianum vs. C. gloeosporioides Weakly pathogenic T. asperellum vs. C. gloeosporioides Weakly pathogenic