Experimental Site
A survey was conducted in four different markets namely Kamal Ranjit market (BAU), Kewatkhali bazaar, Shesh mor market (BAU) and Natun bazaar in Mymensingh district. The clinical experiment was carried out in the Mycology lab of Seed Pathology Centre (SPC), Bangladesh Agricultural University (BAU), Mymensingh during July to December 2007.
Survey on the storage loss of marketed onion due to the diseases
A preliminary survey was done on the stored onion bulbs in above four markets. Four local varieties of onion namely Taherpuri, Faridpuri, Kalashnagari, Zitka and one Indian variety Pusa red were selected for the study. There was no systemic record of the actual storage loss. The losses were mainly due to black mould rot, Fusarium bulb rot and blue mould rot in the markets. To calculate loss, the vendors were motivated to keep purchase and sale records of onion separately. Three kilogram (3 kg) of variety Pusa red and 2 Kg of Taherpuri, Faridpuri, Kalashnagari & Zitka were collected from 4 different markets in Mymensingh town. The samples were drawn at random and placed in separate labeled jute bag. The composite samples were brought to Mycology lab of the Seed Pathology Centre (SPC), BAU, Mymensingh and stored for one week at room temperature (20-29°C). After one week of storage, sample was spread on a working table and the onion bulbs were sorted out into five categories viz. i. Healthy looking bulb (no any symptom/sign on the bulb) ii. Black mould rot symptom bearing bulb iii. Blue mould rot symptom bearing bulbs iv. Fusarium rot symptom bearing bulbs and v. others (having other symptoms/sign). The total number of onion and the number of onion in each category were counted and weighed. All these exercise were done on the basis of direct dry inspection without or with the aid of a 5x hand lens. The difference between the sum of weights of the sorted onions after a week of storage and the weight of the onion bulb sample during collection was loss in weight of onion bulbs. The percent disease incidence was calculated using following equation:
% Disease incidence = No. of diseased ÷ Total no. of onion x 100
Isolation technique
Tissue plating method was used for isolation of associated microorganisms. Diseased onion bulbs were selected for isolation. The selected diseased onion bulb was washed thoroughly to remove soil and sand particles. The infected onion bulb was cut pieces into 5mm in length containing diseased area along with healthy tissues. These inocula were then washed with sterile water for 3-4 times and then placed on the filter paper to remove excess water. Half of the inocula pieces were subjected to surface sterilization by dipping for 5 seconds in 1:1000 HgCl2 solutions. Surface sterilized inocula were then thoroughly (3 times) washed with sterile water and dried on sterile filter paper. Acidified Potato Dextrose Agar (PDA) was used for isolation of associated mycoflora of onion. Two drops of 50% lactic acid was used for 15-20 ml PDA in each 9 cm diameter petridish for acidifying the PDA media to get rid of bacterial contamination. Five cut pieces were placed at equal distances in each of the petridish. Then the plates were incubated in an incubator at 25°C for 3-10 days to allow the pathogen to grow out of the diseased tissues on the medium. Different fungi appearing from the incubated tissues were aseptically transferred to fresh plates containing PDA aiming at culturing one fungal species in separate petridish. Pure culture of individual fungus was prepared through repeated transfer of hyphal tips or single spores. Stock cultures of the isolates were maintained on slants of PDA in test tubes. Most of the associated microorganisms were detected by observing their growth characters on the incubated diseased onion on PDA. Temporary slides were prepared from the fungal colony and observed under compound microscope and identified.
Pathogenicity test
Inoculation of bulbs with injury
Healthy and disease free onion bulbs of all varieties were selected for inoculation. The bulbs were pricked just near the stem with a sterilized needle. The inocula were placed in to the pricked part of the bulbs. Ten healthy bulbs were inoculated where every 2 bulbs were inoculated with Aspergillus niger, A. flavus, Penicillium spp., Fusarium moniliforme and F. oxysporum, respectively maintaining 4 replications with appropriate controls. The test onion was then incubated at 22-23°C for up to 90 days.
Inoculation of bulbs without injury
The same inoculation process (described above) was followed. In this case healthy and disease free onion bulbs were not pricked, just inoculum was placed on to the surface of the necked bulbs. After that test onions were then incubated at 22-23°C for up to 90 days. Disease development was recorded by eye estimation at 15 days intervals.
Experimental design and statistical analysis
The experiment was laid out in a Completely Randomized Design (CRD) with four replications. The data were analyzed using MSTAT statistical package programme. The level of significance and analysis of variance along with the Least Significance Difference (LSD) were done. Mean separation was done by Duncan’s Multiple Range Test (DMRT).