EFFECT OF BIOFUNGICIDE ON THE PRODUCTION OF HEALTHY AND QUALITY SEEDS OF SALVIA HISPANICA IN BANGLADESH

Authors: Md. Rasheduzzaman Azad 1, Md. Zahangir Alam 2, Md. Alamgir Hossain 3 and Md. Atiqur Rahman Khokon 4

Abstract

Experiments were conducted both in the field and laboratory to evaluate the roles of Trichoderma-based biofungicide for improving the health and quality of chia seeds. Four treatments viz. Control-untreated, Seeds treated with BAU-Biofungicide @ 2% of seed weight, Soil treated with IPM lab Biopesticide @ 64kg/ha and Seeds treated with BAU-Biofungicide @ 2% of seed weight + Soil treated with IPM-Biopesticide @ 64kg/ha were used in the field experiment. Among the treatments, BAU- Biofungicide showed better performance in terms of germination percentage, no. of branches, plant height, and seed yield. Both BAU-Biofungicide and BAU-Biofungicide + IPM lab Biopesticide performed better in terms of germination percentage on blotter paper. The identified seed borne fungi were Fusarium oxysporum, Alternaria brassicae and Botrytis cinerea. The prevalence of these fungi was lowest in the seeds treated with BAU-Biofungicide compared to control. Considering storage duration, freshly harvested seeds showed the highest prevalence of seed borne fungi. The quality of chia seeds treated with BAU- Biofungicide remained satisfactory up to six months compared to control. In in-vitro antagonism assay, Trichoderma based BAU-Biofungicide showed best performance to suppress the radial mycelial growth of Fusarium oxysporum and Alternaria brassicae. Application of Trichoderma based BAU-Biofungicide might be effective for growing healthy seeds of chia, as well as seeds can be stored up to six months.

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INTRODUCTION

 

Chia (Salvia hispanica L.) is an annual herbaceous plant of Lamiaceae family. The cultivation of chia is gaining popularity across the world because it is considered as a good source of nutritional and healthy food (Ayerza and Coates 2011). Because of its nutritional value and stability, chia is already added to a range of foods (Ayerza and Coates 2002). Chia seed is consumed as a source of energy, composed of protein (15–25%), fats (30–33%), carbohydrates (26–41%), high dietary fiber (18–30%), ash (4-5%), minerals, vitamins, and dry matter (90–93%). It also contains a high amount of antioxidants (Ixtaina et al. 2008). The seed contains from 25% to 40% oil with 60% (omega) ω-3 alpha-linolenic acid and 20% of (omega) ω-6 linoleic acid of the oil contents (Ayerza and Coates 2011). These two essential fatty acids cannot be synthesized artificially. Both essential fatty acids are required by the human body for good health. However, feeding chia to chickens enriches their meat with omega-3 and to cattle, it enriches their milk with omega-3 (Ayerza and Coates 2002). Recently, chia has been reported as a promising health beneficiary new crop in Bangladesh (Hossain and Fakir 2014). Chia can also be added to commercially prepared infant formulas, baby foods, baked goods, nutrition bars, yogurt and other foods.

Like other plants, chia is also exposed to a number of plant pathogens. The previously reported organisms identified in chia seeds by moist blotter paper and agar plate method were Fusarium solani, Pallidoroseum sp., Rhizopus sp., Cladosporium sp. and the suspected disease is Fusarium wilt. This was evidenced that some plants were found wilted in the field plot (Yeboah et al. 2014).
Still, chia is not cultivated in Bangladesh. But there is a very promising domestic and international market for this crop. Dr. Md. Alamgir Hossain, Professor of the Department of Crop Botany, Bangladesh Agricultural University introduced chia in Bangladesh in 2010. As it is newly introducing crop, the identification of pathogens associated with these seeds and their biological management is urgent for getting seed certification as well as spreading for cultivation in the whole country. Although experiments on plant characters, nutrient content, growth, and production of chia have been done worldwide, disease related to this crop has not been well investigated across the world. So, the present work was undertaken to investigate the effect of Trichoderma based bio-fungicide in improving seed health and quality of chia seeds.Chia seeds are consumed freshly incorporating with yogurt and beverages. However, it is also known that essential oils in chia leaf have repellent properties against insects, making it suitable for organic cultivation. Therefore, organic production is necessary and will get more market value. Biological control of plant pathogens by microorganisms has been considered a more natural and environmentally acceptable alternative to the existing chemical treatment methods (Shalini and Kotasthane 2007; Eziashi et al. 2007; Tumpa et al. 2016). The potential of the antagonistic microorganisms in reducing the intensity of crop damage by the soil-borne plant pathogens has also been reported by Lewis and Larkin, (1997). Several strains of Trichoderma spp. have been found to be effective as biocontrol agents against various soil-borne plant pathogenic fungi such as Rhizoctonia solani, Sclerotium rolfsii, Pythium aphanidermatium, Fusarium oxysporum, F. culmorum and Gaeumannomyces graminis var. tritici and nematodes under greenhouse and field conditions (Cook and Baker 1983, Sivan and Chet 1993, Chet and Baker 1981, Papavizas 1985). Trichoderma strains isolated from Bangladesh and formulated as IPM Lab. Biopesticide showed a broad spectrum of antifungal action against Fusarium oxysporum, Rhizoctonia solani, Fusarium circinatum, Phomopsis vexans, Sclerotium rolfsii and Pythium aphanidermatum (Islam et al. 2016). Moreover, BAU-Biofungicide, a Trichoderma-based biofungicide has been reported for controlling seed-borne, soil-borne and air-borne diseases of different crop plants (Hossain and sultana 2011). Trichoderma inhibits the plant pathogens by a number of ways: mycoparasitism and antibiotic production, competition for nutrient and space with the plant pathogens, production of enzymes such as chitinases and/or glucanases that are responsible for suppression of the plant pathogen. These enzymes function by breaking down the polysaccharides, chitin, and glucans that are responsible for the rigidity of fungal cell walls, thereby destroying cell wall integrity, induction of defense response, and metabolism of germination stimulant (Howell 2003)

 

MATERIALS AND METHODS


In-vitro
antifungal bioassays with BAU Biofungicide and IPM lab Biopesticide were done following dual culture method.  The biofungicides were   evaluated   against Fusarium oxysporum, and Alternaria brassicae by dual culture technique as described by Nafiza (2010).To investigate the effect of Trichoderma based biofungicide on the field performance of chia, seeds of chia were treated with BAU-Biofungicide @ 2% of seed weight, soil treated with IPM lab Biopesticide @ 64 kg/ha. All the necessary intercultural operations were done as and when necessary. After harvesting the seeds were used to investigate the association of seed borne fungi. To investigate the association of seed-borne fungi in `chia’ seeds, seed health testing was done following the standard rules of ISTA (ISTA 2007). The associated pathogens were detected by observing their growth characters on the incubated seeds on blotter paper following the keys outlined by Ramnath et al. (1970), and Khan and Islam (1975). For accurate identification of fungi, temporary slides were prepared from the fungal colony and observed under a compound microscope and identified with the help of keys suggested by Malone and Muskette (1964), Booth (1971), Ellis (1971), and Neergard (1979).The pure culture of the fungus was obtained by culturing the fungus on  PDA  medium  and  making  the fresh culture from  “hyphal  tip”  selected  from  the  periphery  of  actively  growing  colony  under aseptic conditions.

Data were recorded on percent germination, no. of branches per plant, plant height, seed yield, percent moisture contents, and the prevalence of seed-borne fungi at different storage period and radial mycelial growth of the associated seed borne fungi in response to Trichoderma based bio-fungicides. The experiment was conducted with Completely Randomized Design (CRD) with three replications. Data were analyzed through a standard computer package statistical procedure MSTAT-C (Gomez and Gomez 1984).

 

RESULTS

 

Role of Trichoderma-based biofungicide on the field performance of chia

Seed treatment with Trichoderma-based BAU Biofungicide significantly increased percent germination, no. of branches per plant, plant height and seed yield compared to control (Table 1).  Maximum germination (90.83%) was observed from BAU-Biofungicide treated seeds (T1). Germination percentage in other treatments was statistically similar. The lowest germination was recorded in untreated control (T0, 88.96%) Maximum no. of branches per plant (15.00) were recorded in T1 treatments where seeds were treated with BAU-Biofungicide @ 2% of seed weight followed  by 14.27, 14.14, 14.00 in T3 (Seeds treated with BAU-Biofungicide @ 2%+ Soil treated with IPM lab Biopesticide @ 64 kg/ha), T2 (Soil treated with IPM lab Biopesticide @ 64 kg/ha) and T0 (Control) treatments, respectively. The minimum no. of branches per plant was recorded in the untreated control treatment (T0).The highest height (119.9 cm) was recorded in T1 (Seeds were treated with BAU-Biofungicide @ 2% of seed weight) treatment. Statistically similar height 118.4 cm and 118.1 cm were observed in T2 (Soil treated with IPM lab Biopesticide @ 64 kg/ha) and T3 (Seeds treated with BAU-Biofungicide @ 2% of seed weight + Soil treated with IPM lab Biopesticide @ 64 kg/ha) treatment. The lowest height (111.3 cm) was recorded in untreated control (T0) treatment.The highest seed yield (1090 Kg) was recorded in T1 (Seeds were treated with BAU- Biofungicide @ 2%) treatment. Comparatively the lowest seed yield (930 Kg) was found in untreated control (T0) treatment. Higher seed yield (1050 kg and 1033 kg) results were also found from T3 (Seeds treated with BAU-Biofungicide @  2%+ Soil treated with IPM lab Biopesticide @ 64 kg/ha) and T2 (Soil treated with IPM lab Biopesticide @ 64 kg/ha) treatments respectively (Table 1). Seed yield in all treatments except control is statistically similar.

 


Table 1: Effects of Trichoderma-based biofungicide on plant characters

Treatments

 

% Germination No. of     branches per plant Plant Height (cm) Seed Yield      (kg/ha)
T0 88.96   b 14.00   b 111.3   b 930    b
T1 90.83  a 15.00  a 119.9  a 1090   a
T2 89.17   b 14.14   b 118.4  a 1033   a
T3 89.58   b 14.27   b 118.1  a 1050   a
LSD0.05 1.16 0.603 6.08 95.64

T0 = Control, T1 =  Seeds treated with BAU-Biofungicide @ 2% of seed weight, T2 = Soil treated with IPM lab Biopesticide @ 64 kg/hectare (ha) and T3 = Seeds treated with BAU-Biofungicide @ 2% of seed weight + Soil treated with IPM lab Biopesticide @ 64kg/ha

 

Role of Trichoderma based biofungicide on percent germination and moisture contents of seeds at different storage period

According to field experiment, the seeds were harvested and kept separately in plastic container for further study. In order to observe the storage ability of chia seeds, the seeds were preserved in plastic container and were kept in room condition for different storage duration. The seeds were taken out for health test time to time. Germination percentage of freshly harvested seeds did not vary in response to different Trichoderma based bio-fungicide. The range of germination percentage in freshly harvested seeds was 12.10 to 13.30. The results indicate the dormancy of freshly harvested seeds (Table 2). More than 80% germination of chia seeds were recorded after 2 month of storage. Among the treatments, the highest (85.90%) germination was found in T1 (Seeds treated with BAU-Biofungicide) and the lowest (80.20%) in control (T0) (Table 2). Germination of chia seeds in T1, T2 and T3 were statistically similar.

After four month of storage, germination of seeds significantly varied in different treatments ranged from 81.30% to 85.40%. Among the treatments, the highest (85.40%) germination was found in T1 (Seeds were treated with BAU- Biofungicide) and lowest (81.30%) in control (T0) (Table 2). Germination of chia seeds of after four month of storage in T1, T2 and T3 were statistically similar.

After six month of storage, germination of seeds significantly varied in different treatments ranged from 80.80% to 84.70%. Among the treatments, the highest (84.70%) germination was found in T1 (Seeds were treated with BAU- Biofungicide) and the lowest (80.80%) in control (T0) (Table 2). Germination of chia seeds of after six month of storage in T0 and T2 are statistically similar. Germination of chia seeds of after six month of storage in T1 and T3 were statistically similar.


Table 2: Effect of Trichoderma based biofungicide on germination percentage and % moisture content of seed at different storage period 

Treatments Freshly harvested 2 (MAS) 4 (MAS) 6 (MAS)
% germination % moisture % germination % moisture % germination % moisture % germination % moisture
T0 12.80 ab 20.13ab 80.20 b 11.23b 81.30b 14.80a 80.80 b 13.50 c
T1 13.30 a 17.83 b 85.90 a 13.46a 85.40 a 14.20c 84.70 a 14.30 a
T2 12.10 b 21.56 a 83.30 a 14.06a 84.20 a 14.70ab 82.50 ab 13.60 bc
T3 12.20 b 21.42 a 84.20 a 14.01a 83.90 a 14.30bc 83.80 a 13.91b
LSD0.05 0.831 2.67 2.61 1.69 2.57 0.453 2.43 0.376

MAS = Month After Storage

T0 = Control, T1 =  Seeds treated with BAU-Biofungicide @ 2% of seed weight, T2 = Soil treated with IPM lab Biopesticide @ 64 kg/ha and T3 = Seeds treated with   BAU-Biofungicide @ 2% of seed weight + Soil treated with IPM lab Biopesticide @ 64kg/ha

 

The moisture content of chia seeds was determined at different storage duration in response to different Trichoderma based biofungicide. In freshly harvested seeds, the moisture content is high which gradually decreased over the storage time. The moisture content of freshly harvested seeds significantly varied in different treatments ranged from 17.83% to 21.56%. Among the treatments, the highest (21.56%) moisture content was found in T2 (Soil treated with IPM lab Biopesticide @ 64kg/ha) and lowest (17.83%) in seeds treated with BAU-Biofungicide (Table 2). The moisture content of freshly harvested chia seeds in T0   and T1 was statistically similar.

After two months of storage, the moisture content of seeds significantly varied in different treatments ranged from 11.23% to 14.06%. Among the treatments, the highest (14.06%) moisture content was found in T2 (Soil treated with IPM lab Biopesticide @ 64kg/ha) and lowest (11.23%) in control (T0) (Table 2). The moisture content of chia seeds after two months of storage in T1, T2, and T3 were statistically similar. After four months of storage, the moisture content of seeds significantly varied in different treatments ranged from 14.20% to 14.80%. Among the treatments, the highest (14.80%) moisture content was found on T0 (control) and the lowest (14.20%) in T1 (Seeds treated with BAU-Biofungicide) (Table 2). The moisture content of chia seeds in T2, T3, and T1, T3 was statistically similar. After six months of storage, the moisture content of seeds significantly varied in different treatments ranged from 13.50% to 14.30%. Among the treatments, the highest (14.30%) moisture content was in T1 (Seeds treated with BAU-Biofungicide) and the lowest (13.50%) in control (T0) (Table 2).  The moisture content of chia seeds in T2 and T3 was statistically similar.

Role of Trichoderma based biofungicide on percent seed borne infection

Prevalence of seed-borne fungi was recorded at different storage time. The identified fungi were Fusarium oxysporum, Alternaria brassicae, and Botrytis cinerea. The percent seed borne infection by Fusarium oxysporum and Botrytis cinerea was significantly decreased in freshly harvested seeds in response to different Trichoderma based biofungicide (Table 3). However, the percent seed borne infection by Alternaria brassicae was decreased in response Trichoderma based biofungicide but it was not significant statistically (Table 3).

 


Table 3:  Role of Trichoderma based biofungicide on prevalence of seed-borne infection at different storage period

Treatments % seed-borne infection
Fusarium oxysporum Alternaria brassicae Botrytis cinerea
0 MAS 2 MAS 4 MAS 6 MAS 0 MAS 2 MAS 4 MAS 6 MAS 0 MAS 2 MAS 4 MAS 6 MAS
T0 3.200 a 3.100 a 2.980 a 2.580 a 5.87 6.100 a 5.790a 3.64 5.350 a 5.200a 5.280a 2.190a
T1 1.600 c 1.700d 1.760 b 1.780 c 5.60 4.200 d 5.300b 3.43 4.270 b 3.960b 3.890b 1.960 b
T2 2.400 b 2.600 b 2.700 a 2.320b 5.67 5.100 b 5.380b 3.48 5.330 a 5.110a 4.410b 2.120a
T3 1.930 c 2.100 c 1.907 b 1.810 c 5.48 4.600 c 5.310b 3.45 4.710 b 4.210b 4.200b 1.990 b
LSD0.05 0.461 0.326 0.297 0.188 0.465 0.342 0.336 0.429 0.453 0.291 0.505 0.103

T0 = Control, T1 =  Seeds treated with BAU-Biofungicide @ 2% of seed weight, T2= Soil treated with IPM lab Biopesticide @ 64 kg/ha and T3= Seeds treated with   BAU-Biofungicide @ 2% of seed weight + Soil treated with IPM lab Biopesticide @ 64kg/ha

After two, four and six months of storage the percent seed-borne infection by Fusarium oxysporum, Alternaria brassicae and Botrytis cinerea were significantly decreased in response to different Trichoderma based biofungicide (Table 3).

Antagonistic effect of Trichoderma based biofungicide against Fusarium  oxysporum and Alternaria brassicae

The effect of Trichoderma based biofungicide on the radial mycelial growth of Fusarium oxysporum and Alternaria brassicae was recorded up to 72h (Table 4). The radial mycelial growth of Fusarium oxysporum was significantly decreased in response to Trichoderma at 24 hrs, 48 hrs, and 72 hrs. After 24h, the growth of all treatments showed a significant difference. At 24h of incubation, the highest mycelial growth was observed in T0 and T2 treatment while the lowest was observed in T1 and T3 treatment (Table 4). At 48h, the highest mycelial growth was recorded in T0 while the lowest growth was recorded in T1 and T3 treatments. At 72h, the highest mycelial growth was observed in T0 while rest of the treatments showed lower mycelial growth (Table 4).

It was observed that all the treatments showed significant effects on mycelial growth of Alternaria brassicae at 24h, 48h, and 72h of an interval (Table 4). The growth of Alternaria brasssicae at the 24h interval against different treatments was significantly different (Table 4). Among the treatments, the highest mycelial growth (10.33mm) of Alternaria brassicae was observed in control treatment. The lowest growth (8.33mm) was observed in T1. At 24h, the initial growth of this fungus is almost similar. The growth of Alternaria brasssicae at the 48h interval against different treatments was significantly different (Table 4). The highest mycelial growth of Alternaria brassicae (22.67mm) was observed in control treatment and the lowest growth (10.33mm) was observed in T1. The growth of the fungus, Alternaria brasssicae at the 72h interval against different treatments was significantly different (Table 4). Among the treatments, the highest mycelial growth of Alternaria brassicae (30.67mm) was observed in T0. Statistically similar growth of the fungus (15.67mm and 14.00mm) was observed in T2 and T3, respectively. The lowest growth (11.00mm) was observed in T1 (Table 4). Among the treatments Trichoderma based BAU biofungicide showed the best performance.

 


Table 4: Effect of Trichoderma based biofungicide on inhibition of radial mycelial growth of Fusarium oxysporum and Alternaria brassicae

Treatments Radial mycelial growth (mm)
24h 48h 72h
F. oxysporum A. brassicae F. oxysporum A. brassicae F. oxysporum A. brassicae
T0 9.670 a 10.330 a 14.67 a 22.67a 18.67 a 30.67a
T1 8.330 b 8.330 c 11.33 c 10.33c 11.67 b 11.00 c
T2 9.330 a 9.290 b 13.00 b 15.67b 12.33 b 15.67b
T3 9.000 b 9.330b 11.67 c 15.33b 12.00 b 14.00b
LSD0.05 0.665 0.854 1.09 1.06 1.11 2.13

T0 (control= No Trichoderma), T1 (Trichoderma harzianum of BAU-Biofungicide), T2 (Trichoderma harzianum of IPM lab Biopesticide) and T3 (Trichoderma harzianum of BAU-Biofungicide + Trichoderma harzianum of IPM lab Biopesticide).

 

 

DISCUSSION

 

Seed treatment and soil treatment with Trichoderma-based BAU Biofungicide significantly increased percent germination, no. of branches per plant, plant height and seed yield compared to control. Abd-El-Khair et al. (2010) reported that Trichoderma spp. improved the plant characters and decreased the damping off disease incidence of the bean. Alam et al. (2014) reported that seed treatment with Trichoderma based biofungicide decreased the prevalence of seed-borne fungi of chili. Sultana et al. (2009) and Hossain and Sultan (2011) reported that BAU-Biofungicide increased germination and seedling vigor of some vegetable seeds. Naznin and Hossain (2005) observed 50.80% higher germination over the control in cowpea by applying BAU-Biofungicide. Trichoderma harzianum treated seed of blackgram resulted up to 16.66% seed germination over control (Shamsuzzaman and Hossain (2003). These results indicate that Trichoderma might suppress the growth of soil-borne pathogens and stimulate the growth of plants.


In this experiment, the efficacy of bioagents against the isolated seed borne pathogens was assessed in lab condition. Trichoderma harzianum of BAU-Biofungicide showed better performance to suppress the growth of all isolated pathogens. Therefore this can be hypothesized that application of bioagents in the field suppressed both soil-inhabiting and seed borne fungi. Moreover, these bioagents may also activate some enzymes in the host plants which suppress the growth of pathogen as well as produce healthy seedlings. The findings of some researchers also support the present findings that Trichoderma-based bioagents can promote vegetative growth and increased yield of different crops. Harman (1990) reported that Trichoderma harzianum provided good control against a   range   of   pathogens,   including   Phytophthora, Pythium   ultimum, Rhizoctonia   solani, Fusarium spp., Sclerotium rolfsii and Botrytis cinera, if properly applied. Xu et al. (1993) found that mycelial growth of Fusarium solani and F. oxysporum was inhibited with T. harzianum. Michalikova and Michrina (1997) reported the greatest inhibition rate of the radial growth of F. culmorum (55-58%) with T. harzianum. Similar findings were obtained by Begum (1997), Sultana et al. (2001), and Kashem (2005). Trichoderma inhibit the plant pathogens by a number of ways: mycoparasitism and antibiotic production, competition for nutrient and space with the plant pathogens, production of enzymes such as chitinases and/or glucanases that are responsible for suppression of the plant pathogen. These enzymes function by breaking down the polysaccharides, chitin, and glucans that are responsible for the rigidity of fungal cell walls, thereby destroying cell wall integrity, induction of defense response, and metabolism of germination stimulant (Howell 2003).

CONCLUSIONS

 

Chia is a promising health beneficial new crop to be released in Bangladesh. The results of this research suggest that Trichoderma based biofungicide improves the field performance of chia, reduces the prevalence of major seed-borne fungi of chia, decreased the radial mycelial growth of seed-borne fungi in-vitro and improves the storability of chia seeds. Since Trichoderma based biofungicide promotes the production of healthy and quality seeds of chia, as well as chia has enormous beneficial effects on health; so it can be introduced in Bangladesh as a new crop. Therefore, Biofungicide can be recommended for better production of chia in Bangladesh.

 

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[1]2017 Bangladesh Phytopathological Society

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1 Graduate Student, 2 Assistant Professor, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, 3 Professor, Department of Crop Botany, Bangladesh Agricultural University, Mymensingh,4 Professor,Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh.

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