Studies on variability in mode of infection by the isolates of leaf pathogens isolated from diseased leaves of Musa paradisiaca from Uttar Dinajpur District

Introduction

Between December 2022 and January 2023, leaf spot disease was observed on leaves of field-grown banana plants in the Uttar Dinajpur District, West Bengal. Diseased leaf samples were collected from Ramganj (Lat - 25.64855° long - 88.430628°), Gotgaon (Lat - 25.646622° long - 20 88.440895°) and Kaluhar ((Lat - 25.637923° long - 88.444335°) places. Symptoms first appeared as yellowish-brown spots in the middle and margin of leaves. Symptoms range from a few lesions scattered across leaves to lesions densely covering large sections of leave (Figure 1).

Figure 1. Collection of diseased leaf samples of banana.

Two fungal isolates - MUSLF-2, MUSLF-3 and one fungal isolate - MUSLF-1 were isolated from the infected leaf from Ramganj and Gotgaon locations onto potato dextrose agar amended with Monocef. Based on light microscopic and scanning electron microscopic studies, MUSLF-2 and MUSLF-3 were identified as Curvularia sp. (Figures 2 & 3). Koch’s postulate was successfully established for Curvularia sp. causing leaf spot disease in banana. Disease symptoms were developed within seven days on inoculated detached leaves and in inoculated leaf surfaces, conidial germination was observed. Curvularia sp. was reisolated from the infected spots. Leaf diseases by Curvularia sp. or Curvularia lunata or other species of Curvularia were reported by previous researchers (Addrah et al., 2021; Manzar et al., 2021) in support of our research findings.

Figure 2. Transfer of diseased leaf samples to media and isolation of leaf pathogens from diseased banana leaves along with variability in their morphologies.

Figure 3. SEM view of MUSLF-2 and MUSLF-3 isolates; Test of pathogenicity and establishing Koch’s Postulates by MUSLF-2 and MUSLF-3 isolates - Curvularia sp. (Spore suspension showing spore count).

Mycelial invasion of the pathogen in infected leaf was confirmed through the section of leaves under light microscope with cotton blue staining (Figures 4 & 5). The surface morphology of healthy and Curvularia sp. inoculated leaves were studied by scanning electron microscopy (Figures 6 & 7). Changes in internal structures of healthy and Curvularia sp. inoculated leaves were observed through transmission electron microscopy (Figure 8). Role of Curvularia in causing leaf spots in other crop plants was studied by researchers (Khoo et al., 2022; Bandara et al., 2022).

Figure 4. Leaf samples after artificial inoculation with spore suspension of MUSLF-2 and MUSLF-3 isolates - Curvularia sp. - 1×10³ conidia/ml following detached leaf technique; Section of Curvularia sp. infected leaf of Musa paradisiaca under light microscope with cotton blue stain.

Figure 5. Section of MUSLF-1 infected leaf of Musa paradisiaca under light microscope with cotton blue stain; Conidial germination observation under light microscope in Curvularia sp. infected leaf of Musa paradisiaca after artificial inoculation in detached leaf technique.

Figure 6. Conidial germination observation under Scanning Electron Microscopy (SEM) in Curvularia sp. infected leaf of Musa paradisiaca after artificial inoculation in detached leaf technique.

Figure 7. Hyphal observation under Scanning Electron Microscopy (SEM) in MUS LF-1infected leaf of Musa paradisiaca after artificial inoculation in detached leaf technique.

Figure 8. Observation of internal structure of healthy and infected leaf of banana under Transmission Electron Microscope (TEM).

After Koch’s postulate, three fungal isolates- MUSLF-2, MUSLF-3 [Curvularia sp.] and MUSLF-1 grown in sand maize meal media were applied in banana field along with foliar spray of spore suspensions (1×10³ conidia/ml). Although the method of inoculation was somewhat artificial, the symptoms were similar to those observed in the in vitro detached leaf inoculation condition (Figures 9 & 10). Similar finding on first report of Curvularia lunata causing leaf spots in banana plant from Uttar Dinajpur District is in support of our present research (Chowhan and Chakraborty, 2022).

Figure 9. Preparation of sand maize meal media; Growth of Curvularia sp. (MUSLF-2, 3) and MUSLF-1 isolate in sand maize meal media.

Figure 10. Application of Curvularia sp. (MUSLF-2, 3) and MUSLF-1 grown sand maize meal media in rhizpspheric zone of banana and foliar spray of spore suspensions in leaves of Musa paradisiacal; Establishment of leaf spot in field and confirmation of Curvularia sp.

In a nutshell, the research finding focuses on the nature of symptoms of leaf spot diseases of banana and role of Curvularia sp. in establishment of leaf spot through Koch’s postulate in detached leaf technique as well as in field condition.