Allium cepa L. (Onion) Storage Diseases and Effect of Trichoderma asperellum and Trichoderma virens Pre-harvest Treatments on Postharvest Quality

Substantial yield losses have been reported during the storage of Allium cepa L., and effective methods to minimize postharvest losses are currently lacking. Therefore the objectives of this study were to investigate the impact of pre-treatment with Trichoderma


INTRODUCTION
Allium cepa L., commonly known as big onion, is utilized as a condiment in many countries worldwide and holds significant economic value as a cash crop in Sri Lanka. Onions thrive in moderate climates, avoiding excessive rainfall and extreme temperatures. Optimal conditions for early growth involve cool temperatures ranging from 20°C to 25°C, alongside sufficient moisture supply. As the plants mature and approach harvest, warm and drier conditions are preferable. The pH requirement falls between 5.8 and 8.0, with the optimum range being 6.0 to 7.0 (Maankotte, 2004;Rathnayaka, 1992). The Agriculture and Environmental Statistics Division of the Department of Census and Statistics in Sri Lanka (2020) reports that big onion cultivation is primarily concentrated in four districts: Matale, Anuradhapura, Mahaweli H and to a lesser extent in Polonnaruwa.
Big onion in Sri Lanka experiences a consistent consumer demand of approximately 200,000 MT throughout the year. However, the annual national production falls significantly short at only about 68,643 MT (Department of Census and Statistics, Sri Lanka, 2021). Consequently, it becomes crucial to increase production and minimize losses caused by various factors.
The objective of storing onion bulbs is to preserve their quality and extend the marketing period. Successful storage of onion bulbs is vital to ensure a year-round supply for consumption and seed production. As storage organs by nature, bulbs are wellsuited for this purpose. Onion dormancy initiates through a decline in respiration and abscisic acid levels within the bulbs. Nonetheless, during storage, challenges arise from issues such as sprouting, drying and rotting, resulting in losses (Coolong, 2007).
Big onions are susceptible to various diseases that can occur at any stage of their growth in the field or during storage. These diseases can be classified into three categories: seedling diseases, foliage diseases and bulb diseases, primarily caused by fungi and bacteria, resulting in significant losses to the harvest. Substantial losses in crop yield result from plant diseases caused by phytopathogenic agents. In recent times, there has been an increasing focus on biological control methods for fungal pathogens as alternatives to the use of chemicals. Synthetic pesticides are expensive, contribute to environmental pollution, and pose potential risks to animals and humans. Furthermore, their repeated application leads to the development of chemically resistant strains of pathogens (Vinale et al., 2014).
One of the most effective strategies for pest management in agriculture is the utilization of microbes in biological control. Biological control involves harnessing the benefits of beneficial organisms, their genes, and/or their products, such as metabolites, to mitigate the negative impacts of harmful organisms, including plant pathogens, while promoting positive interactions with desirable organisms like crops, beneficial insects, and microorganisms (Pal and Gardner, 2006).

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While commercial formulations of Trichoderma are available, it is advisable to utilize locally isolated Trichoderma spp. as biocontrol agents, as they cause minimal disruption to the soil's microflora. Previous efforts have been made to develop an efficient formulation of locally isolated Trichoderma spp. for biocontrol against damping off disease and basal rot of onions , Gunaratna et al., 2020 (b)). Based on the literature, two methods, seed coating and soil inoculation, were tested for introducing the prepared Trichoderma inoculum under greenhouse conditions. The results indicated that seed priming and soil treatment with Trichoderma spp. were suitable for field trials. Previous studies demonstrated the efficacy of inocula prepared from two locally isolated Trichoderma spp. in significantly reducing the incidence and severity of damping off and basal rot diseases in A. cepa L., both under greenhouse and field conditions, when applied individually or in combination. Therefore, the prepared inoculum could effectively control damping off and basal rot diseases of A. cepa L. in the field. Furthermore, treatment with Trichoderma spp. enhanced the plant height, dry weight, and fresh weight of tested A. cepa L. seedlings and transplants (Gunaratna et al., 2020 (b)). Based on the above findings, the objectives of this study were formulated to evaluate the effect of pretreatment with Trichoderma spp. on disease control during the storage of Allium cepa L., identify factors contributing to storage losses of Allium cepa L., identify fungi associated with storage deterioration of Allium cepa L., and identify fungi associated with stored bulb tissue samples of Allium cepa L. after different seed pre-treatments in Sri Lanka.

Effect of pre-harvest treatments to control diseases during storage
Seeds of the onion cultivar "Galewela light red" were sown into seed beds in May 2015 and grown for four weeks. In the nursery trial, twelve treatments were tested, which included Talc-based formulations of Trichoderma asperellum and T. virens ). In June 2015, the seedlings were uprooted and transplanted into raised beds. During the transplanted stage, eleven treatments were tested, including the aforementioned Talc-based Trichoderma spp. formulations (Gunaratna et al., 2020 (b)). In September 2015, the plants were manually harvested, stubbed, and had their leaves mowed, while stones and clods were removed. The bulbs were then cured at 32°C for 3 days under ambient drying conditions, with careful attention given to avoiding physical injury to the bulbs during these operations.

Storage
Seventy-five (75) randomly selected healthy bulbs harvested from different pre-treated fields were stored according to a complete randomized design with three replications. The onions were stored in open-mesh packages at a temperature and relative humidity of approximately 30-32°C and 77-79%, respectively. During storage, there were four categories: bulbs neither pre-treated with Trichoderma spp. nor fungicides, bulbs pre-treated with Trichoderma asperellum, bulbs pre-treated with Trichoderma virens and Trichoderma asperellum, and bulbs pretreated with fungicides (Mancozeb, Homai, Captan).
At two-week intervals, the bulbs were turned, and the number of diseased bulbs was recorded. After removing the bulbs from storage bags, they were weighed, cut longitudinally, and visually assessed for disease symptoms. Diseased bulbs were carefully separated and recorded to avoid contaminating the healthy bulbs. The final number of bulbs on the last data collection date was subtracted from the initial number of bulbs, and the difference was calculated as the percentage lost due to diseases. The weights of healthy bulbs were measured at the end of the 14 th week period to determine the storage losses during this period. The disease status in each category was calculated using the following formula.

Determination of weight loss during storage
The weight loss during storage was calculated, after removing the dry scales at the end of the storage period using the following formula.
( ) ( -) Isolation and identification of fungi associated with diseased big onion bulbs during storage The diseased onions during storage were sorted out based on macro symptoms of infection, and these samples were pooled together according to their macro-symptoms. Infected scales of the onion bulbs measuring 1 cm x 1 cm were removed using a sterile scalpel blade and surface sterilized with 70% ethanol for one minute. The pieces were then rinsed three times with sterilized distilled water and dried on a sterile filter paper. The edges of the sections were trimmed, and pieces measuring 0.5 mm x 0.5 mm were plated under aseptic conditions on Potato Dextrose Agar medium (PDA) supplemented with Tetracycline (PDA+Tet.). The plates were incubated at room temperature for seven days. Similarly, the infected outer dry scales of onion bulbs showing black mold were removed with a sterile scalpel and directly plated on PDA+Tetracycline plates, which were then incubated at room temperature for seven days. The fungal isolates were identified based on their macro and micro-morphological characteristics using the sticky tape method. The frequency of occurrence of each fungal species was calculated using the following formula:

Isolation of associated fungi from roots / leaves of sprouted onion bulbs during the storage
Fungal isolation was conducted using the tissue plating method described above. Root pieces measuring 1 cm in length and leaf pieces measuring 5 mm x 5 mm (from roots and leaves of onions pre-treated with Trichoderma spp., onions neither pre-treated with Trichoderma spp. nor fungicides, and onions pre-treated with fungicides) were placed on PDA+Tet. medium and incubated at room temperature for seven days. The fungal isolates were identified based on their macro and micro-morphological characteristics, using the sticky tape method.

Seed infestation test (Agar Plate Method)
Twenty-five non-surface sterilized A. cepa L. seeds were placed in petri plates containing Potato Dextrose Agar medium (PDA) supplemented with tetracycline. The plates were then incubated at room temperature for seven days. The isolated fungal genera were identified based on their macro and micromorphological characteristics, using the sticky tape method.

Blotter method
The blotter method was utilized to detect fungi associated with the surface of A. cepa L. seeds. In this method, two layers of sterile Watman No. 1 filter paper were moistened with sterilized water and placed at the bottom of a 15 cm diameter petri dish. Batches of 40 non-sterilized seeds were then placed on the moist filter paper. After plating, the seeds were incubated at room temperature for seven days. At the conclusion of the incubation period, the fungi were identified based on observed characteristics of the colony and microscopic structures.

Effect of pre-harvest treatments on control of diseases during the storage
Two post-harvest fungal storage diseases were identified as black mold and basal rot based on previously available data (Table 1). Furthermore, a certain level of storage loss was attributed to the sprouting of onion bulbs. The study revealed that bulb diseases associated with fungi accounted for approximately 33.33% to 65.33% of the losses during storage.
The results of percent diseased bulbs indicated that there was no significant effect of pre-harvest treatments, whether pretreated with Trichoderma spp. or pre-treated with fungicides, on the control of diseases during storage ( Table 2).

Determination of weight loss during the storage
The average percent weight loss during storage ranged from 6.26% to 34.53%.

Isolation and identification of fungi associated with diseased big onion bulbs during storage
a) Percentage occurrence of isolated fungi associated with basal rot disease of A. cepa L. during the storage Mucor sp. had the highest frequency of occurrence (50%) among the surfacesterilized infected scales of the onion bulbs, followed by Fusarium sp. (44%). Penicillium sp. recorded the lowest frequency of occurrence (6%). b) Percentage occurrence of isolated fungi associated with black mould disease of A. cepa L. during storage Aspergillus niger had the highest frequency of occurrence (44.4%), followed by Aspergillus flavus (40.0%), among the black mould disease-bearing non-surface sterilized dry scales. Penicillium sp. had a frequency of occurrence of 6.7%. Fusarium sp. and Mucor sp. had the lowest frequency of isolation, both at 4.4%.

Isolation of associated fungi from roots/leaves of sprouted onion bulbs during the storage
The following fungal genera were isolated by tissue plating method (Table 3).

Determination of seed quality a) Seed infestation test (Agar Plate Method)
The fungal pathogens isolated from the A. cepa L. seeds were Aspergillus spp., Curvularia spp., Alternaria sp. , Fusarium sp. b) Blotter method Aspergillus niger was isolated from nonsurface sterilized seeds using Blotter method.   Big onions are an essential condiment in the daily diet of Sri Lankans, with a consistent year-round demand totaling approximately 200,000 tonnes annually (Customs report, 2009). The production of big onions is limited to a seasonal cultivation period from April to September, known as the yala season, with harvesting taking place in September and October. However, the off-season shortage and restricted cultivation result in the importation of big onions to meet the demand.
Proper preharvest and postharvest conditions are essential for the storability of onion bulbs, as they play a significant role in ensuring a fresh market supply and availability for processing purposes (Adamicki, 2005). Additionally, Allium cepa L. seeds play a crucial role in disseminating and developing diseases during different growth stages. Seed-borne diseases can also be transmitted through seeds, causing damage at various stages of Allium cepa L. growth, from germination to crop maturity, and even during storage, resulting in substantial losses (Dumbre et al., 2011). Therefore, determining seed quality is equally important.
The current study revealed that the surfaces of Allium cepa L. seeds were contaminated with Aspergillus niger and other internal contaminants, including Aspergillus spp., Curvularia spp., Alternaria sp., and Fusarium sp., using the Agar plate method, similar to the work conducted by Dumbre et al., in 2011. In their study, Dumbre et al., (2011 identified 12 different fungi, including five species of Aspergillus, two species of Fusarium, and one each from Penicillium, Drechslera, Curvularia, Rhizopus, and Alternaria. Among the various fungi, Aspergillus niger was found to be dominant in Allium cepa L. seeds collected from different locations in India, with a frequency of 58.88% in non-surface treated seeds and 33.55% in seeds treated with HgCl2. Further, Koycii and Ozer (1997) reports, high frequency of Aspergillus and also Nagerabi and Abdalla (2004) established that Aspergillus was the most prevalent genus among all the seed borne fungi of onion. The findings of Adongo et al., (2015) reported that A. niger was the most frequently encountered fungus from the seeds of Bawku Red onion variety. A. flavus, A. niger were also isolated by Jidda and Benjamin (2016) from seed samples of Monguno Red onion variety. Additionally, Alternaria alternate, Alternaria porri, Rhizopus stolonifer, Botrytis allii and Penicillium roqueforti, Fusarium spp. were also isolated from onion seeds other than Aspergillus spp. (Adongo et al., 2015;Jidda and Benjamin 2016).
During the current study, two major diseases were encountered during the storage period: black mould disease and basal rot disease. In addition to diseases, storage losses occurred due to sprouting and rooting of Allium cepa L. bulbs. Similar findings were reported by Currah and Proctor (1990), who identified sprouting and bulb rotting as major causes of losses. The present study revealed that five fungal genera were associated with postharvest storage diseases: A. niger, A. flavus, Fusarium sp., Mucor sp., and Penicillium sp. These findings are consistent with those reported by Jidda and Benjamin (2016), who also isolated A. niger and Fusarium sp. from rotten red onion bulbs.
| 167 Adongo et al., (2015) conducted a survey of fungi associated with postharvest deterioration of onion bulbs and found that rotten onion bulbs were infected by Aspergillus niger, A. flavus, Penicillium sp., Rhizopus stolonifer, and Fusarium oxysporum. Major storage rots identified included black mould, blue mould, soft rot, neck rot, and basal plate rot. Similar to the current findings, black mould was the most predominant storage disease. Mahmud and Monjil (2015) also reported that losses were mainly due to black mould rot and Fusarium bulb rot. Similarly, Ko et al., (2002) stated that black mould caused by A. niger is the major disease during storage under ambient conditions in the tropics.
Seeds are considered to be sources responsible for the transmission of pathogens. The presence of Aspergillus spp. and Fusarium sp. in seed samples, as well as in black mould and basal rot diseased bulbs during storage, suggests the possibility of fungal transmission through seeds, which later act as agents of storage diseases. The Food and Agriculture Organization of the United Nations has indicated that A. niger infection occurs during onion production, but the development of the fungus on the bulbs takes place during storage when the environmental conditions are conducive to their growth. Furthermore, the results revealed that preharvest treatments, such as pre-treatment with Trichoderma spp. or fungicides, did not have a significant effect on disease control during storage. However, standard fungicide treatments during field conditions reduced the percentage of diseased bulbs after harvest (40%), compared to control treatment (56.44%) or pre-treatments with Trichoderma spp., such as pre-treated with Trichoderma asperellum (51.55%) and pretreated with Trichoderma virens and Trichoderma asperellum (48.89%). Nevertheless, Trichoderma virens and Trichoderma asperellum, isolated from the soils of A. cepa L. growing areas and applied as seed coating, seedling root dip, or soil inoculation, could be effectively used in the management of Allium cepa L. damping off and basal rot diseases under greenhouse and field conditions , Gunaratna et al., 2020(b)).
Additionally, some onions were spoiled due to sprouting. Suojala (2001) reported that storage losses were primarily caused by sprouting, rooting, and wilting. The prevalent temperature (~30-32°C) and relative humidity (~77%-79%) during the storage period might have enhanced sprouting. Appropriate storage temperature and relative humidity can help prevent sprouting. According to the Food and Agriculture Organization of the United Nations, storage at temperatures of 25-30°C has been shown to reduce sprouting and root growth compared to low-temperature storage (10-20°C). However, high-temperature storage can lead to weight loss, bulb desiccation, and rot. Successful low-temperature storage requires proper ventilation and a relative humidity range of 70-75%. However, for tropical climates, the organization recommends hightemperature storage with proper ventilation, temperatures above 25°C, and humidity levels of 75-85%.
Moreover, during post-harvest, fungal genera such as Fusarium sp. and Mucor sp. were found to be associated with the root tissues of sprouted onion bulbs during storage. Moreover, Aspergillus sp. was isolated from the root tissues of sprouted onion bulbs that were pre-treated with Trichoderma spp., while only Mucor sp. was isolated from the root tissues of sprouted onion bulbs pretreated with fungicides. Aspergillus sp. was also isolated from the leaf tissues of sprouted onion bulbs that underwent pre-treatment with Trichoderma spp., whereas Fusarium sp., Penicillium sp., and Mucor sp. were isolated from the leaf tissues of sprouted onion bulbs that were neither pre-treated with Trichoderma spp. nor fungicides.

CONCLUSIONS
Five fungal genera, namely Aspergillus niger, Aspergillus flavus, Fusarium sp., Mucor sp., and Penicillium sp., were found to be associated with post-harvest storage diseases in Allium cepa L. Additionally, the major contaminants of Allium cepa L. seeds were identified as Aspergillus spp. The main postharvest diseases observed in Allium cepa L. were black mould disease and basal rot disease. Furthermore, significant losses were attributed to sprouting, and some weight loss was also reported during storage. It was determined that neither pre-harvest treatments with Trichoderma spp. nor fungicides were effective in controlling diseases during the storage of Allium cepa L.