A synopsis of research proposal

Biocontrol of Anthracnose of Chilli by Using Endophytic Bacterial

Consortia

INTRODUCTION
Chilli (Capsicum annum L.) is an important spice in Bangladesh which belongs to the family
Solanaceae. It is found as green, dried and powdered forms. It is primarily used in every food for
its pungency, taste, colour and aroma. Chilli contains high amount of beta- carotene, capsaicin
and vitamin A, C; provitamin E, P, B1 B2 and B3 (Reddy 2023). In Bangladesh, chilli is usually
cultivated during both Rabi and Kharif seasons. In Kharif, 50291.18 acres produce128602.24
MT and 192709.69 acres produce 496223.53 MT in Rabi, respectively (BBS. 2022).
Several factors viz. lack of high-yielding cultivar, low seed quality, diseases, and pests are
responsible for the reduced yield. Among all of these limitations, Anthracnose disease caused by
Colletotrichum spp. is one of the most severe and important causes of yield losses in chili pepper
ranging from 10 to 80% (Asare-Badiako et al. 2015). Colletotrichum gloeosporioides is one of
the important pathogens causing anthracnose disease in chili, which can cause yield losses of up
to 90% (Gautam et al. 2012). Anthracnose disease control commonly carried out in the field
involves the intensive use of pesticides. Intensive and unwise use of chemicals (pesticides) may
leave toxic effects in the environment, and the breeding resistant genes have no long-lasting
durability (Ji et al. 2008), leading to a risk for public health, natural waters, and non-target
organisms (Gikas et al. 2022). Thus, other environmentally friendly alternatives are needed, one
of which is the using biological agents from the group of endophytic bacteria (Yanti et al. 2020).
Objective
1. To develop formulated bio-fungicide using Bacterial consortium
2. To observe the efficacy of formulated bio-fungicide against fusarium wilt of chilli in field
conditions
3.To observe shelf-life of the formulated bio-fungicide
Material and methods
Experimental site: The experiment will be conducted in the laboratory and research field under
the Department of plant Pathology, HSTU, Dinajpur.
Isolation of fungal pathogen: Surface sterilized (0.5% NaOCl) vascular tissues from the
infected plants will be placed on moist blotter papers in Petri dishes and incubated at 25±2 °C for
7 days in the dark. The isolated fungi will be identified morphologically using a compound
microscope.
Collection of beneficial bacteria: Two beneficial bacteria viz. Serratia marcescens and
Rhizobium have already been isolated and one of them have been identified on the basis of
molecular characterization.
Confirmation of endophytic bacteria:
KOH test: Gram test was carried out by dripping 3% KOH solution on the slide surface, then a
single colony of endophytic bacteria was placed. If there is mucus when the needle is removed,
the bacteria are Gram-negative, while if there is no mucus, the bacteria are Gram-positive
(Schaad et al. 2001).
Pathogenicity Test of C. gloeosporioides: The pathogenicity test was performed using healthy
chilies. Suspension of C. gloeosporioides (106 conidia/mL) was inoculated by smearing with a
brush on the injured part. The fruit was placed in a plastic box filled with a moist paper tissue
and observed until symptoms appeared. Symptoms are shown by lesions on the infected fruit
(Ibrahim et al. 2017).
Ability test of the endophytic bacterial consortium to control anthracnose disease in chili
Disease development
Incubation period (days after inoculation): The incubation period was observed every day
after the inoculation of C. gloeosporioides until the plants showed early anthracnose symptoms
in chili. The initial symptoms of C. gloeosporioides are characterized by the presence of round,
grooved brown spots. The effectiveness of suppression of the incubation period was calculated
using the formula (Sivan and Chet 1986)
T −nc
E= × 100
nc
Where:
E: effectiveness
T: the value of treatment
nc: the value of Negative control
Collection of chilli seeds and raising of seedlings: Chilli seeds (BARI Morich-1 and BARI
Morich-4) will be collected from Bangladesh Agricultural Research Institute (BARI), Rajbari,
Dinajpur. Collected seeds will be treated with selected beneficial bacteria.
Field experiment: Field trials will be carried out following randomized complete block design
(RCBD) with three replications. The following treatment combinations will be applied in field
conditions viz.
T0: Control; T1: Charcoal-based formulated Rhizobium sp.+ S. marcescens; T2:Wood dust-
based formulated Rhizobium sp.+S. marcescens; T3:Talc powder- based formulated Rhizobium
 sp.+S. marcescens; T4: Pea bran-based formulated Rhizobium sp.+S. marcescens;
T5:Vermicompost-based formulated Rhizobium sp.+S. marcescens; T6:Commercial bacterial
formulation ; T7:Vulcan(Carbendazim 50wp @0.5g/L water).
Collection of data: Data will be collected on seed germination (%); percent wilt disease;
severity of wilt disease; plant height (cm); number of branches, number of flowers; number of
fruits, fruit setting, shoot length, shoot weight, root length, root weight, yield (kg), ascorbic acid
content, total soluble solids, firmness, total phenol content and different mineral.
Statistical analysis: The obtained data will be statistically analysed using Statistix10 software of
analysing of variance.
Expected Outcome: After successful completion of the experiment, we will be able to reduce
the use of chemical with the increased chilli yield.
Socio Economic Importance: Farmer mostly depends on chemicals to control plant diseases. To
offer the alternative option rather than chemical control, bio-agents like bacteria could be a
better candidate to boost the eco-friendly management of fusarium diseases. However, using
bacterial formulation may replace the costly chemical by inducing resistance in plants which
ultimately will offer safe and healthy environment with reducing the loss of yield.

Tentative Schedule

Activities Activity performance schedule during the project period

Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug

Collection of review of
literature

Isolation and identification

of the fungal pathogens

Collection of beneficial
bacteria

Raising of seedling

Preparation of formulated
bacteria

Shelf-Life Study
Field experiment

Data collection

Biochemical analysis

Statistical analysis

Thesis writing

References

Asare-Badiako E, Addo-Quaye A, Boakye B, Sarbah JM, Asante P, Dorm E. 2015. Incidence

and severity of viral and fungal diseases of chili pepper (Capsicum frutescens) in some
districts in Chana. Intl J Plant Soil Sci 1: 147-159 DOI: 10.9734/IJPSS/2015/16830.
BBS. (2022). Yearbook of agricultural statistics, Bangladesh Bureau of Statistics Ministry of
Planning, Govt. of Bangladesh, Dhaka, Bangladesh.Pp.24-36.
Gautam AK, Avasthi S, Bhadauria R. 2012. First report of Colletotrichum gloeosporioides on
Pedilanthus tithymaloides in India. J New Biol Rep 1 (1): 3-5.
Gikas GD, Parlakidis P, Mavropoulos T, Vryzas Z. 2022. Particularities of fungicides and factors
affecting their fate and removal efficacy: A review. Sustainability 14: 4056. DOI:
10.3390/su14074056.
Ibrahim R, Hidayat SH, Widodo. 2017. Morphology, genetic, and pathogenicity variability of
Coletotrichum acutatum the causal agent of anthracnose on chilli in Java and Sumatera.
Jurnal Fitopatologi Indonesia 1 (13): 9-16. DOI: /10.14692/jfi.13.1.9. [Indonesian]
Ji GH, Wei LF, He YQ, Wu YP, Bai XH. 2008. Biological control of rice bacterial blight by
Lysobacter antibioticus strain 13-1. Biol Cont 45: 288-296. DOI:
10.1016/j.biocontrol.2008.01.004.
Reddy, K.M. (2023). Capsicums for Nutrition and Entrepreneurship. In Vegetables for Nutrition
and Entrepreneurship. pp. 297-309. Singapore: Springer Nature Singapore.
Schaad NW, Jones JB, Chun W. 2001. Laboratory Guide for Identification of Plant. Pathogenic
Bacteria. The American Phytopatology Society, St Paul.
Sivan A, Chet I. 1986. Biological control of Fusarium spp. in cotton, wheat and musk melon by
Trichoderma harzianum. J Phytopathol 116: 39-47. DOI: 10.1111/j.1439-
0434.1986.tb00892.x.
Yanti Y, Hamid H, Reflin, Warnita, Habazar T. 2020. The ability of indigenous Bacillus spp.
consortia to control the anthracnose disease (Colletotrichum capsici) and increase the growth
of chili. Biodiversitas 21 (1): 179-186. DOI: 10.13057/biodiv/d210123.

________ _________
Signature of the Student Signature of the Supervisor
Name: Chamak Sarker Dr. Md. Mohidul Hasan
M.S in Plant Pathology, Professor, Department of Plant Pathology
Department of Plant Pathology, Faculty of Agriculture
Faculty of Agriculture Hajee Mohammad Danesh Science &
Hajee Mohammad Danesh Science & Technology University, Dinajpur-5200
Technology University, Dinajpur-5200 Mobile: 01718545630
Mobile: 01796794958 Email: mhasan@hstu.ac.bd
Email: chamaksarker99@gmail.com