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The Diversity and Antagonistic Ability of Trichoderma spp. on the Aspergillus Flavus Pathogen on Peanuts in North Center of Vietnam

Nguyen Thi Thanh*, Ho Thi Nhung, Nguyen Thi Thuy, Thai Thi Ngoc Lam, Phan Thi Giang, Tran Ngoc Lan, Nguyen Van Viet, Vu Trieu Man

Vinh University, Vinh, Vietnam

Abstract: Among 1000 peanut soil samples were collected from Nghe An province, Thanh Hoa province and Ha Tinh province, 44.90% samples occurred Trichoderma. The appearance ratio of Trichoderma in Nghe An, Thanh Hoa, Ha Tinh is 44.30%, 52.00% and 39.50%, respectively. By using in the dual culture method on PDA, The Percent Inhibition of Mycelial Growth (PIRG) was determined to be very high in 28 Trichoderma strains (made up 28.3% in total), 29 others strains was dertermined that to be high PIRG (made up 29,3% in total). The highest PIRG was 100%, occurred on T. harzianum (Tri.019(4).NC; Tri.053(1).TG; Tri.011(1).NL; Tri.002(2).NX strain), T. atroviride (Tri.020(2).NC; Tri.011(1).NC strain), T. reesci (Tri.007(1).NĐ), T. hamatum (Tri.039(1).TG), T. virens (Tri.014(4).NC) and T. pseudokonigii (Tri.014(1).NL).

Keywords: Antagonistic ability, Aspergillus flavus, Diversity, PIRG, Trichoderma

Received September 09, 2014; Revised November 10, 2014; Accepted December 19, 2014

Citation: Nguyen Thi Thanh, Ho Thi Nhung, Nguyen Thi Thuy, Thai Thi Ngoc Lam, Phan Thi Giang, Tran Ngoc Lan, Nguyen Van Viet, Vu Trieu Man, The Diversity and Antagonistic Ability of Trichoderma spp. on the Aspergillus Flavus Pathogen on Peanuts in North Center of Vietnam. World Journal of Agricultural Research, 2 (6). 291-295. January 2014. doi:10.12691/wjar-2-6-8

1. Introduction

In Vietnam, peanut (Arachis hypogaea L.) is known as a short- day crop of high economic value, to be used as food and for export. Peanut contains 50% lipid, 22-25 % protein, and also contains 8 essential amino acids and soluble vitamins such as B1 (Thiamine), B2 (Riboflavin), PP (nicotinic oxide), E F ... (Nguyen Van Binh et al.). Peanut was listed among top ten exported products of Vietnam. It was the second biggest exported crops after rice of Vietnam (Vietnam General Statistics Office, 2004). Simultaneously, peanut exportation reached 32000 – 35000 ton, ranked 5th out of ten biggest export countries of the world. In 2013, peanut cultivated surface of Vietnam was approximately 216300 ha, its production reached to 492600 ton.

North Center region of Vietnam, includes three provinces Thanh Hoa, Nghe An and Ha Tinh, has the largest cultivated surface of peanut, make up 23.30% in total cultivated surface and 22.91% of total production in whole country. Cultivated surfaces of Nghe An, Ha Tinh, Thanh Hoa were 19600 ha; 17300 ha; 13500 ha, made up 9.06%; 8.00%; 6.24% in total peanut cultivated surface of Vietnam, respectively. Peanut productions of these provinces were 44500 ton, 40800 ton, 27600 ton, respectively made up 9.03%; 8.28%; 5.60% in total production (Vietnam General Statistics Office, 2013).

Aspergillus flavus pathogen mainly infect peanut, produce toxic compound called aflatoxin, causes health risks like cancer and other dangerous diseases in human and animals. Many countries have attempted to limit exposure to aflatoxins by imposing regulatory limits on importation of peanut. To help minimize risk for human and animal health, many cultivate peanut countries, include Vietnam, have concerned in researching and controlling A. flavus fungi on peanut.

In recent years, researchers on the world tend to use biological control to prevent Aspergillus flavus fungi causing aflatoxin by three main ways: (i) Using Bacillus (B. pumilus, B. subtilis) (Munimbazi et al., 1998); (ii) Using nontoxigenic Aspergillus flavus, A. Parasiticus strains, that had successful application in US, like Aspergillus flavus AF36 (Cotty et al., 2004); (iii) Using antagonistic fungi Trichoderma, also had good result, such as using five antagonistic fungi species T. harzianum, T. viride, T. auroviride, T. longibrachiatum, T. hamatum on bio-control Aspergillus flavus causing aflatoxin in India (Mausam et al., 2007; Reddy et al., 2010).

In Vietnam, famers still mainly use cultivations practices and some storage methods based on their experience to management aflatoxin produced by A. flavus fungi on peanut, such as control crop season, using lime, expose peanut under the sun to ensure the humidity less than 9%, and store peanut in cool and dry condition. Scientists have researched methods of bio-control and resistant variety production to prevent A. flavus fungi causing aflatoxin. In 2010, Nguyen Thuy Chau et al. had first success on coffee, maize and peanut in not only pre- but also post-harvest stage by using nontoxigenic A. flavus fungi. Up to now, in Vietnam we have determined at least thirty three species of Trichoderma. But the application of antagonistic fungi Trichoderma to management aflatoxin produced by Aspergillus flavus in agriculture is still not concerned.

Because of above reasons, we carry out the research: The diversity and antagonistic ability of Trichoderma spp. on the Aspergillus flavus pathogen on peanuts in north center of Vietnam.

2. Materials and Methods

2.1. Materials

- 1000 soil samples were collected from peanut cultivation regions of Nghe An, Thanh Hoa, Ha Tinh provinces, Vietnam.

- Trichoderma strains: Eight species of Trichoderma -T. harzianum, T. atroviride, T. reesci, T. hamatum, T. virens, T. pseudokonigii, T. aureoviride, T. viride were isolated from different soil samples, cultured on PDA (Potato Dextro Agar) medium. Five days old culture of Trichoderma was used for each experiment.

- Pathogen source: Aspergillus flavus (VAD006) was isolated from the infected peanut seeds and cultured on PDA medium. Seven days old culture of pathogen was used for each experiment.

- Culture mediums: Water Agar (GA), Potato Dextrose Agar (PDA).

2.2. Methods

2.2.1. Collection and Isolation

- Collecting soil samples was followed by Lester, 2009 [2].

- Isolating Trichoderma from the soil different was followed by Waksman (1952); Johnson et al., (1962).

Test the antagonistic ability of Trichoderma on A. flavus: Evaluation the antagonistic ability of Trichoderma on A. flavus was followed by Rahman, M. F. Begum and M. F. Alam. (2009). In this method, the 90mm diameter culture plates contain Potato Dextro Agar (PDA) medium were used. On the medium of dual control plate, Trichoderma (T) was placed 2 cm away from the edge of the petri plate. A. flavus (C) was similarly placed 2 cm away from the edge of the petri dish and on the opposite side with Trichoderma sample. On a control plate, A. flavus was placed with the same manner on a fresh PDA plate (Figure 1). All pairings were incubated at 30°C. Antagonistic ability was tested after 7 days of incubation by measuring the radius of the A. flavus colony in the direction of the antagonist colony (R₂) and the radius of the A. flavus colony in the control plate (R₁). All the data was processed by using the formula developed by Skidmore and Dickinson (1976) - Percentage inhibition of radial growth (PIRG) formla.

Figure 1. Measurement of radial growth of A. flavus mycelia by method where culture placement was 2 cm away from the margin. Note: R1, radius of A. flavus colony in control plate; R2, radius of A. flavus colony in dual culture plate; C, A. flavus isolate; T, Trichoderma isolate

R1: The radius of A. flavus mycelium in the control plate

R2: The radius of A. flavus mycelium in the dual culture plate (Trichoderma and A. flavus).

The antagonistic levels:

PIRG ≤ 50%: Low

50% < PIRG ≤ 60%: Medium

60% < PIRG ≤ 75%: High

PIRG >75%: Very high

3. Results and Discussion

3.1. Collection, Isolation of Trichoderma Strains From the Soil Grows Peanuts in North Center Vietnam

Evaluation the occurrence frequency of the fungus Trichoderma over 1000 peanut soil samples in 3 representative provinces of the North center as showed in Table 1.

Table 1. The occurrence frequency of Trichoderma in North Center of Vietnam, 2011-2013

Through the collection of 1000 soil samples from three provinces, the appear frequency of Trichoderma strains was 44.90%. The appear frequency of Trichoderma strains of Nghe An, Thanh Hoa, Ha Tinh was 44.30%, 52.00% and 39.50%, respectively. In Nghe An province, the occurrence frequency of Trichoderma in Dien Chau district was the highest with 55.00%, Quynh Luu district with 49.50% and the lowest was in Nghi Loc district with 28.50%.

3.2. Evaluating the Antagonistic Ability of Trichoderma spp. on A. Flavus Pathogen on Peanuts in North Center Vietnam

3.2.1. The Antagonistic Ability of T. harzianum on A. flavus

The antagonistic ability of 21 T. harzianum strains on A. flavus was shown at Table 2.

Table 2. The antagonistic ability of T. harzianum strains on A. flavus after 7 days of old fungal culture

Among 21 T. harzianum strains, 6 strains were showed at very high level of antagonistic ability included Tri.019(4).NC, Tri.053(1).TG, Tri.002(2).NC, Tri.011(1).NL, Tri.002(2).NX and Tri.004(2).NX. There were 8 strains (38.09%) were showed at high antagonistic level.

3.2.2. The Antagonistic Ability of T. atroviride on A. flavus

The antagonistic ability of 18 strains of T. atroviride and A. flavus showed at Table 3.

Table 3. The antagonistic ability of T. atroviride on A. flavus after 7 days of old fungal culture

Eight strains (44.44%) were showed at very high PIRG among 18 strains of T. atroviride, including Tri.020(2).NC, Tri.011(1).NC, Tri.005(2).NĐ, Tri.009(1).NC, Tri.041(1).NĐ, Tri.005(1).NX, Tri.008(1).NT and Tri.038(2).NX. There were 3 strains (16.67%) with PIRG at high level.

3.2.3. The Antagonistic Ability of T. reesci on A. flavus

The antagonistic ability of 10 strains of T. reesci on A. flavus were showed at Table 4.

Table 4. The antagonistic ability of T. reesci on A. flavus after 7 days of old fungal culture

The antagonistic level (20.00%) were very high in 2 strains Tri.069(2).QX and Tri.007(1).NĐ. There were 2 strains (20.00%) showed with PIRG at high level.

3.2.4. The Antagonistic Ability of T. hamatum on A. flavus

The antagonistic ability of 20 strains of T. hamatum on A. flavus showed at Table 5.

Table 5. The antagonistic ability of T. hamatum on A. flavus after 7 days old fungal culture

Among 20 strains of T. hamatum that were inoculated, the PIRG of 5 strains (25.00%) were show at very high including Tri.039(1).TG, Tri.149(1).TG, Tri.095(1).TH, Tri.092(1).TH and Tri.008(1).NC. There were 8 strains (40.00%) with PIRG at high level.

3.2.5. The Antagonistic Ability of T. virens on A. flavus

The antagonistic ability of 20 strains on T. virens and A. flavus showed at Table 6.

Table 6. The antagonistic ability of T. virens on A. flavus after 7 days of old fungal culture

Result showed that, there were four strains (20.00%) that very high antagonistic level and 6 strains (30.00%) at high level.

3.2.6. The Antagonistic Ability of T. pseudokonigii, T. aureoviride, T. viride on A. flavus

Table 7. The antagonistic ability of T. pseudokonigii, T. aureo viride, T. viride on A. flavus after 7 days of old fungal culture

T. pseudokonigii had 3 strains which showed at very high antagonistic level including Tri.013(1).NC, Tri.014(1).NL, Tri.006(2).NX. T. aureoviride had 2 strains with the high antagonistic level. The PIRG of T. viride was 38.4%, showed at low antagonistic level with A. flavus.

Figure 2. The antagonistic ability of Trichoderma spp. on A. flavus: a. The radius of A. flavus mycelium after 7 of inoculation in the control plate, b-i: The radius of A. flavus mycelium in the dual culture plate, shows overgrowth of Trichoderma covering the A. flavus colony after 7 days of inoculation in dual culture: T. harzianum (Tri.011(1).NL, Tri.002(2).NC, Tri.053(1).TG), T. atroviride (Tri.005(1)NX, Tri.011(1).NC, Tri.020(2).NC, T. hamatum (Tri.092(1).TH), T. virens (Tri.046(2).NĐ), respectively

4. Conclusion and Recommendations

4.1. Conclusions

(1) Among 1000 soil samples that cultivate peanuts from three provinces Nghe An, Thanh Hoa, Ha Tinh, the appear frequency of Trichoderma strains was 44.90%. The appear frequency of Trichoderma strains of each province was: Nghe An 44.30%; Thanh Hoa 52.00% and Ha Tinh 39.50%.

(2) Evaluation the antagonistic ability of 99 Trichoderma strains on A. flavus was carried out by using dual old fungal culture method on PDA medium. There were 28 strains (28.3%) showed at very high antagonistic level. The highest PIRG was 100% included T. harzianum (Tri.019(4).NC; Tri.053(1).TG; Tri.011(1).NL; Tri.002(2).NX strain), T. atroviride (Tri.020(2).NC; Tri.011(1).NC strain), T. reesci Tri.007(1).NĐ, T. hamatum Tri.039(1).TG, T. virens Tri. 014(4).NC and T. pseudokonigii Tri.014(1).NL.

4.2. Recommendations

Using Trichoderma strains that have the very high antagonistic level on Aspergillus flavus for further research to make bio-products of control Aspergillus flavus.

Acknowledgements

To make the contents of this article, the authors would like to thank the Ministry of Science and Technology of Vietnam for financial support with project's code: ĐTĐL.2011G/28.

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