In Vitro Antagonistic Study of Maize Root Colonizing Fungal Isolates Against Fusarium moniliforme Causing Ear Rot Disease of Maize

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2021, Vol. 11, No. 2, 133 – 139 http://dx.doi.org/10.11594/jtls.11.02.02

How to cite:

Tiru Z, Sarkar M, Chakraborty AP et al. (2021) In Vitro Antagonistic Study of Maize Root Colonizing Fungal Isolates Against Research Article

In Vitro Antagonistic Study of Maize Root Colonizing Fungal Isolates Against Fusarium moniliforme Causing Ear Rot Disease of Maize

Zerald Tiru, Monalisha Sarkar, Arka Pratim Chakraborty, Ayon Pal, Parimal Mandal^*

Mycology and Plant Pathology Laboratory, Department of Botany, Raiganj University, Raiganj, Uttar Dinajpur, Pin-733134, India

Article history:

Submission October 2020 Revised October 2020 Accepted February 2021

ABSTRACT

In the present study, different root colonizing fungal isolates were isolated from the rhizospheric soil of maize growing areas of Uttar Dinajpur, West Bengal. All the isolates including test pathogen were identified from Indian Type Culture Collection, New Delhi. Plant growth promoting maize root colonizing fungal isolates- Penicillium pinophilum (ITC NO. 11,201.19), Trichoderma harzianum (ITC NO.11,203.19), Trichoderma asperellum (ITC NO. 11,209.19), Aspergillus niger (ITC NO.

11,204.19) and Penicillum purpurogenum (ITC NO. 11,207.19) exhibited antagonistic activities against F. moniliforme (ITC NO. 11,208.19) in vitro. Two antagonistic isolates of T. harzianum and eleven strains of T. asperellum showed antibiosis mechanism for antagonism against F. moniliforme with the range of Percent Inhibition of Radial Growth from 62.41% to 88.57%. Competitive mode of antagonism against test pathogen by the isolates of P. pinophilum, P. purpurogenum and six isolates of A.

niger were found. Percent of inhibition of radial growth ranged from 57.14% to 91.42%. In our finding, antagonistic isolates especially Trichoderma harzianum (ITC NO. 11,203.19), Trichoderma asperellum (ITC NO. 11,209.19) strains either single or in combination with rest maize root colonizing strains could be used as potent growth promoters as well as biocontrol (BCA) agents.

Keywords: Ear rot disease of maize, Fusarium moniliforme, In vitro antagonism- Competition/Antibiosis, Maize rhizosphere, Root colonizing plant growth promoting fungi

*Corresponding author:

E-mail: [email protected]

Introduction

Maize (Zea mays L.) is the 2^nd major cereal cash crop after rice at Uttar Dinajpur, West Ben- gal, India. It hosts many pathogenic microorganisms that impart diseases and damages to the plant and negatively affect the yield and quality of the seed. Fusarium ear rot has been considered one of the most destructive bio-agent as it is responsible for yield loss and affects the quality of seeds of maize [1]. Still, it is not easy to control fully, even by many commonly available synthetic fungicides. Despite all these negative impacts of causal organisms on maize plants, it still remains one of the preferable choices of many maize growers in this region. Indiscriminate use of chemical fungicides to minimize the pathogen impact poses a se- rious threat to soil health, may reduce soil-

inhabiting beneficial microbial population and give space for development of fungicide resistant pathogen. Many spp. of Trichoderma [2, 3] have been reported to have a significant role as biofer- tilizer because of their root colonizing ability, growth promotional activities and enhancing the many defensive tactics against pathogens [4].

Presently for management of ear rot disease of maize, bio-control agent (BCA) is getting much more attention for sustainable agriculture as it is considered as economically viable because of its effectiveness, not causing environmental and health hazards. Antagonistic microorganisms are widely used as bio-fungicides in sustainable agriculture.

BCA performs antagonistic activity for a specific pathogenic microorganism by mechanisms such

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JTLS | Journal of Tropical Life Science 134 Volume 11 | Number 2 | May | 2021

as competition, antibiosis, mycoparasitism and endophytic growth etc.

In competition, BCA may grow faster and utilize food sources more efficiently at its niche than the soil borne pathogenic microorganism, thereby re- duction of the population of soil borne pathogenic microorganism. The most common reason for the death of many microorganisms growing in the vi- cinity of BCA is the limiting sources of nutrients [5, 6].

For antibiosis, antagonistic microorganisms may release some antimicrobial compound that slows down or completely inhibit the growth of pathogenic microorganisms. There were several reports by researchers about the antibiosis mechanisms adopted by antagonistic microbes like Tricho- derma against strains of Fusarium [7, 8].

In case of mycoparasitism, BCA may feed on pathogenic microbes directly. Mycoparasitism is one of the main mechanisms of Trichoderma, reported by previous researchers who focused the production of extra cellular enzymes, chitinase by Trichoderma isolates that would help in inhibition of other pathogens [9, 10].

Endophytic growth: Endophytic activity of many microorganisms may useful to host plant by stim- ulating of plant growth promotional and defense activities. They are present inside the different plant organs and activate production of bioactive compound to boost defense mechanism. As per reports, there are many endophytic microorganisms like Trichderma and Penicillium which colonise the root of host plant and produces growth promoting ingredient and enhance the defense system in many plants [11, 12].

The present study was undertaken to screen indigenous BCA found in rhizosphere of maize growing region of Uttar Dinajpur, West Bengal, India and to know their antagonistic efficacy against Fusarium moniliforme causing ear rot disease of maize.

Material and Methods

Collection of soil sample from maize rhizosphere Rhizospheric soil samples were collected from maize growing region of Uttar Dinajpur, West Bengal, India. The plant was gently and carefully uprooted; soil tightly adhered to root was collected

in sterile poly bag and brought to the Mycology and Plant Pathology Laboratory, Raiganj Univer- sity.

Isolation of maize root colonizing fungal isolates Collected soil samples were air dried for 4 hr and isolation was done by serial dilution technique [13]. Potato dextrose agar (PDA) medium was used to isolate different root colonizing fungal isolates and screen their antagonistic activity against causal pathogen causing ear rot disease of maize.

1 ml soil suspension in the range of 10^-4 to 10^-5 was taken with the help of 5 ml sterilized pipette, poured on the petri plate and sealed with parafilm.

The plates were incubated at 28±1^oC for 3-7 days.

Observation on the appearance of any colonies was recorded from 3^rd to 5^th day. Individual colonies were picked up and maintained in pure culture for further study.

Isolation of causal pathogen of ear rot disease of maize

The pathogenic isolate was isolated from ear rot infection of maize. A diseased part of the plant was collected from the field; the surface was sterilized with a 0.1% mercuric chloride solution, washed three times thoroughly with the distilled sterilized water, placed on PDA plates aseptically, and incubated at 28 ± 1°C.

In vitro antagonistic activity of root colonizing fungal isolates against ear rot pathogen

Dual culture assay was performed to screen antagonistic agents against maize ear rot pathogenic fungus, following the method of Skidmore and Dickinson (1976) with slight modification [14]. A mycelial disc (5 mm in diameter) of pathogenic fungal mycelia from the 7day old culture was cut using sterile cock borer, placed it at a distance of 2 cm from the edge on petri plate having PDA and a disc of 2 mm root colonizing fungal isolate of 7 days old culture was also cut, placed towards the other edge at the same distance on the same petri plate. In control, sterile distilled water was added in place of root colonizing fungal isolates. The plates were kept under incubation at 28

± 1°C and observed daily until the growth of the fungal pathogen reached towards the edge of the plate in the control plate. Mycelial growths of the fungal pathogen on both the plates were measured and Percent Inhibition of Radial Growth (PIRG) was calculated using the formula developed by

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Skidmore and Dickinson (1976) [14]:

Percent Inhibition of Radial Growth (PIRG)

=(𝑅₁− 𝑅₂) 𝑅₁ × 100

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R1= Diameters (cm) of pathogenic fungal mycelia in the control plate

R2= Diameters (cm) of the pathogenic fungal mycelia in the treated plates

The tests were carried out in triplicates and repeated twice.

Results and Discussions

Morphological observation and Identification of root colonizing fungal isolates and causal patho- gen of ear rot

Twenty-three different maize root colonizing fungal isolates and causal fungal pathogen of ear rot of maize were isolated from maize field of Raiganj, Kaliaganj, Itahar and Hemtabad area of Uttar Dinajpur. Those isolates were identified from the Indian Type Culture Collection, New Delhi as Penicillium pinophilum (ITC NO.

11,201.19), Trichoderma harzianum (ITC NO.

11,203.19), Trichoderma asperellum (ITC NO.

Table 1. Survey, collection of Root Colonizing Fungal Isolates (RCFI) of maize rhizosphere, pathogen causing ear rot disease of maize in maize filed, located at Uttar Dinajpur, West Bengal and morphological identification of the isolates and pathogen from the Indian Type Culture Collection, New Delhi Mycoflora isolates

(Root Colonizing Fungal Isolates (RCFI) of maize rhizosphere)

Place of collection with isolate codes No. of

isolates ITC No.*

Raiganj Kaliaganj Itahar Hemtabad

Penicillium pinophilum AA AA1 - - 2 11, 201.19

Trichoderma harzianum CC CC1 - - 2 11, 203.19

Aspergillus niger RGG1,

RGG2, RGG3

RGG4, RGG5,

RGG6 - - 6 11,204.19

Penicillum purpurogenum RGC1 - - RGC2 2 11,207.19

Trichoderma asperellum

RGH1, RGH2, RGH3, RGH4, RGH5, RGH6

-

RGD2, RGD3, RGD4, RGD5, RGD6, RGD7

- 12 11,209.19

Fungal Pathogen of maize rhizosphere

Fusarium moniliforme RGE1 - - - 1 11,208.19

Total numbers of isolates including root colonizing fungal isolates along with one

fungal pathogen 24+1

* ITC Number of Indian Type Culture Collection

Table 2. Morphological and microscopic characteristics of Root Colonizing Fungal Isolates (RCFI) of maize rhizosphere and pathogen causing ear rot disease of maize

Mycoflora isolates Morphological and microscopic characteristics Aspergillus niger

(ITC NO. 11,204.19) Colonies: black on PDA media; Mycelia: hyaline, aseptate; Conidia:

Conidial heads radiate, conidia brown, ornamented with warts and ridges, subspherical; Conidiophore: Consisting of a dense felt of conidiophores. conidiophore stipes smooth walled, hyaline, vesicles sub spherical.

Trichoderma asperellum (ITC NO. 11,209.19)

Colonies: dark green; Conidia: subglobose to ovoidal, long, smooth and green in color.

Penicillium purpurogenum (ITC NO. 11,207.19)

Colonies: olive green; Mycelia: Hyaline, aseptate; Conidia: smooth- walled, ellipsoidal to cylindrical, 6-9 in number

Fungal Pathogen of maize rhizosphere Fusarium moniliforme

(ITC NO. 11,208.19) Hyphae are septate and hyaline, conidiophores are medium length, microconidia abundant, oval to clavate.

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11,209.19), Aspergillus niger (ITC NO.

11,204.19), Penicillum purpurogenum (ITC NO.

11,207.19) and ear rot fungal pathogen as F. mo- niliforme (ITC NO. 11,208.19) (Table 1). Fungal cultures were maintained on PDA slants at 4°C for further study. Morphological features of fungal

antagonistic isolates A. niger, T. asperellum, P.

purpurogenum along with test pathogen F. monil- iforme were examined under bright field micro- scope (Figure 1). The morphological and microscopic features of the isolates were recorded (Ta- ble 2).

Figure 1. Microscopic observation of root colonizing fungal antagonistic isolates: A = A. niger (ITC NO.

11,204.19); B= T. asperellum (ITC NO. 11,209.19); C= P. purpurogenum (ITC NO. 11,207.19) along with test pathogen: D = F. moniliforme (ITC NO. 11,208.19)

Table 3. Antagonistic activity of different root colonizing fungal isolates (RCFI) against Fusarium monili- forme

Root Colonizing Fungal Isolates

(RCFI) with isolate codes* Paired with test pathogen

code (RGE1) ** % of Inhibition of radial

growth*** Mechanism of antagonism AA

RGE1

91.42±2.91 Competition

AA1 82.85±2.66 Competition

CC 77.14±2.89 Antibiosis

CC1 71.42±2.82 Antibiosis

RGH1 82.85±2.66 Antibiosis

RGG1 86.85±2.33 Competition

RGD2 67.71±2.10 Antibiosis

RGC1 88.57±2.67 Competition

RGC2 69.79±1.89 Competition

Note:

***% of Inhibition of radial growth observed after 7 days; ** Test pathogen code (RGE1)- Fusarium moniliforme (ITC NO. 11,208.19);* Root Colonizing Fungal Isolates (RCFI)- AA, AA1 = Penicillium pinophilum (ITC NO.

11,201.19); CC, CC1= Trichoderma harzianum (ITC NO. 11,203.19);RGH1, RGH2, RGH3, RGH4 & RGH5=

Trichoderma asperellum (ITC NO. 11,209.19); RGG1, RGG2, RGG3, RGG4, RGG5 & RGG6= Aspergillus niger (ITC NO. 11,204.19); RGD2, RGD3, RGD4, RGD5, RGD6 & RGD7= Trichoderma asperellum (ITC NO.

11,209.19); RGC1, RGC2= Penicillum purpurogenum (ITC NO. 11,207.19).

±- SE; Average of three replicates.

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Figure 2. In vitro antagonistic tests of Root Colonizing plant growth promoting fungal isolates (RCPGPF), isolated from the rhizosphere of Z. mays- AA, AA1 = P. pinophilum (ITC NO. 11,201.19); CC, CC1=

T. harzianum (ITC NO. 11,203.19);RGH1, RGH2, RGH3, RGH4 & RGH5= T. asperellum (ITC NO.

11,209.19); RGG1, RGG2, RGG3, RGG4, RGG5 & RGG6= A. niger (ITC NO. 11,204.19); RGD2, RGD3, RGD4, RGD5, RGD6 & RGD7= T. asperellum (ITC NO. 11,209.19); RGC1, RGC2= P.

purpurogenum (ITC NO. 11,207.19) against test pathogen- F. moniliforme (ITC NO. 11,208.19).

Code (RGE1)- Respective control plate of F. moniliforme.

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In support of morphological identification of our isolated fungal pathogen- F. moniliforme, there were previous reports by scientist about de- tailed study of taxonomy and biology of F. monil- iforme [15]. Several researchers also studied about the taxonomy of T. asperellum strain Ta13, isolates of A. niger and P. purpurogenum GH2 [16, 17, 18].

Screening of in vitro antagonistic activity of an- tagonistic fungal isolates against F. moniliforme

Out of twenty-three different maize rhizospheric root colonizing fungal isolates tested for their antagonistic efficacy against F. moniliforme, thirteen of them showed antibiosis with the range of Percent Inhibition of Radial Growth (PIRG) from 62.41% to 88.57% with treatment of different T. asperellum isolates. Other ten rhizospheric isolates of P. pinophilum and A. niger showed competition against F. moniliforme with range of PIRG from 57.14% to 91.42% (Table 3; Figure 2).

Antagonistic potential of T. harzianum was also observed by Gwa and Nwankiti [19] in his research finding against F. moniliforme. Highest (58.70%) mean percentage growth inhibition was observed when T. harzianum was applied as an- tagonist before inoculation of F. moniliforme, whereas least percentage growth inhibition (34.33%) was found after application of T. harzi- anum+ F. moniliforme. In another study, antagonistic activities of Trichoderma strains against Fusarium oxysporum f. sp. melongenae were observed [20]. T. harzianum showed highest antagonistic efficiency (81.11%) and Trichoderma kon- ingii exhibited least antagonistic efficacy of 28.88% against brinjal vascular wilt causing pathogen.

There were reports about the role of many plant growth promoting phosphate solubilizing antagonistic fungal isolates in controlling several pathogenic strains of microorganisms [21]. There had been numerous reports on the ability of Trichodema species to antagonize a wide range of commercially important plant pathogens [22, 23].

Antagonistic roles of Trichoderma and Glio- cladium against Fusarium udum were found as per the findings made by Pandey and Upadhyay [24].

Similarly, combination of different strains of Trichoderma, Gliocladium virens and Aspergillus nidulans were most effective to control Rhi- zoctonia solani [25, 26]. Antagonistic activities of different isolates of Aspergillus against Fusarium

were reported by Hidayat et al. [27]. It was found that Aspergillus sp. Strains- PD2, PD4 and PD5 inhibited the pathogen growth by 37.31%, 26.52%

and 12.04% respectively. Roles of Trichoderma viride, T. harzianum and T. koningii as biocontrol agents against seed-borne pathogenic fungi like F.

moniliforme, Curvularia lunata were reported by Raihan [28].

Conclusion

The overall results of the present study depicts that plant growth promoting maize root colonizing fungal isolates have shown antagonistic activity against Fusarium moniliforme, causal fungal pathogen of ear rot disease of maize plant. In our finding, such antagonistic isolates especially Tricho- derma harzianum (ITC NO. 11,203.19), Tricho- derma asperellum (ITC NO. 11,209.19) strains, isolated from maize rhizosphere, either single or in combination with rest maize root colonizing strains could be used as potent growth promoters as well as biocontrol (BCA) agents against fungal pathogens which may pave the way towards eco- friendly sustainable agriculture by reducing environmental hazards.

Acknowledgement

Authors are grateful to the team of Division of Plant Pathology, IARI, New Delhi for authentic identification of Root Colonizing Plant Growth Promoting Fungal isolates and fungal pathogen of maize.

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