Bacterial Rot of Paddy Straw Mushroom and its Management

Mushrooms are basically fungi, which have a fleshy and spore-bearing fruiting body Verma et al., (2017). The key elements of the global mushroom industry are edible and medicinal mushrooms. Vegetable flesh is another name for edible mushrooms Jaiswal et al., (2024). There are about 2000 mushroom species in nature, but only about 25 are commonly used as food (Chatterjee and Patel, 2016). The countrys old tradition of cultivated mushrooms originated in China (Boa, 2004). Mushroom production worldwide was increased to 10,378,163 metric tons in 2016. Currently, countries with the highest mushroom production are China, USA, Poland, Netherlands, India, France, Spain, Canada, Mexico etc.
       
Medicinal or edible mushrooms are often recognized as nutritious foods. Nutraceuticals confirm to attract the growers and researchers attention. The global mushroom production has increased more than 27-fold in the last 35 years from approximately 1 billion kg in 1978 to approximately 27 billion kg in 2012 which is remarkable especially in comparison to the global human population which has increased 1.7-fold in the same duration from approximately 4.2 billion in 1978 to approximately 7 billion in 2012 Royse, (2014). According to Royse et al., (2017), people currently consume 5 kg of mushrooms per year on average. In particular, proteins, minerals and vitamins as well as bioactive substances like steroids, polysaccharides and phenolic compounds are abundant in mushrooms Okhuoya et al., (2010). It is the best food choice for patients with heart disease and high blood pressure because of its high potassium-to-salt ratio. According to Nongthombam et al., (2021), a variety of metabolites found in mushroom species have been shown to exhibit anticancer, antioxidant, antigen toxic, antiplatelet aggregating, antihyperglycemic, antimicrobial and antiviral effects. In India Punjab, Haryana, Uttar Pradesh, Rajasthan, Jammu and Kashmir and Odisha are the major mushroom-producing states. From 2010-17, the mushroom industry in India have reported an average growth of 4.3% per annum. Out of total mushroom produced, the major contributors are white button (73%) followed by oyster (60%), paddy straw (7%) and milky mushroom (3%). Mushrooms as compared to vegetables, per capita consumption of mushroom is meager and data indicates that it is less than 100g/year in 2016-17. India’s mushroom industry increased by exporting 1054 quintals of white button mushrooms as reported by Sharma et al., (2017).
       
Mushrooms with their medicinal, nutritional and savory qualities are seen as the future of vegetables in today’s health-conscious society. They offer high digestibility and abundant protein making them a potential meat substitute Pavel (2009). In addition, they are a fantastic source of vitamin D which is not found in other food supplements Pehrsson et al., (2003). Vitamin D and protein deficiencies are amongst the most prevalent malnutrition problems of India and several other developing nations Srivastava et al., (2023). Mushrooms like all other crops are impacted by a variety of biotic and abiotic factors. The production of mushrooms is vulnerable to a variety of competing organisms and weed fungus Brown (1937); Davis (1938). Nematodes, bacteria and other pollutants have been documented to harm mushrooms both directly and indirectly. During the period of incubation of the spawn and cultivation of the oyster mushroom Pleurotus ostreatus, Trichoderma harzianum, Coprinus spp., Aspergillus niger  and Penicillium spp. were collected Akhter et al., (2017) which caused specific diseases in mushroom sporophores. A large no of other fungi grows within the mushroom bed as competitors significantly affecting the yield of mushrooms. Many fungal diseases can affect commercial mushroom crops Fletcher and Gaze, (2008). Similarly, few viral diseases have been reported to be associated with edible mushrooms Sharma, (2017); Sharma et al., (2007). However, bacteria are considered as major threat to mushroom production. Many bacterial diseases have been reported from all over the world in the fruit bodies of A. bisporus, A.bitorquis, Pleurotus spp., Volvariella sp., Lentiluna edodes, Flamulina velutipes and Auricularia spp. According to Fermor, (1986), losses from mushroom spoilage caused by the bacterial blotch (Pseudomonas tolaasii) are estimated to be 5–10% of the total mushrooms cultivated. On the other hand, the traditional cultivation methodology of Volvariella sp. provides enough scope for development and colonization of mushroom mycelium and fruiting bodies by wide range of bacterial pathogen as prevalence of high moisture and warm temperature provides ideal ecological condition for colonization of bacterial pathogen within the mushroom substrate. The research on bacterial pathogens is limited to bacterial rot of Volvariella sp. caused by Pseudomonas sp. Kannaiyan, (1978).
       
Lack of research on occurrence of bacterial diseases of Volvariella sp. led to deterioration in marketable mushrooms quality and storability. The present study was carried out to explore the possibilities of bacterial pathogens responsible for causing rotting of marketable mushrooms of Volvariella sp.

The experiment was carried out in the Department of Plant Pathology, Institute of Agricultural Sciences, SOADU, Bhubaneswar in the year 2021. The infected samples were collected from different sources and mushrooms showing water-soaked lesion developed at the tip of the vulva which enlarged and extended towards the base of the fruiting body were selected. The infected area was sunken as it became thin. The internal tissues resembled yellow to light brown in color. As the disease advanced, the vulva became slimy and sunken circular to irregular spot developed on the cap or pileus of the mushroom. Below the cap the gills looked water-soaked and in advance case of infection the gills were disintegrated and these symptoms were recorded (Fig 1). Bacteria associated with symptom category were isolated. Apparently, infected mushrooms were selected for isolation of the bacteria for the purpose. Each mushroom was surface disinfected by cleaning the mushroom surface with a cotton swab soaked in 70% ethyl alcohol. The surface disinfected mushrooms were longitudinally cut by a surgical knife sterilized by alcohol. A five-millimeter size tissue from the diseased area was cutoff and placed in a drop of sterile water for about 15-20 minutes or till the water became turbid. A loop full of bacterial suspension was taken and cross-streaked on the dry surface of a nutrient agar plate for the isolation of bacteria. The plates were kept in an upside-down position in a BOD incubator maintained at 27±1º c for 24-48 hours for the development of bacterial colonies. One or two frequently growing colonies having distinct colony characters were selected. A selected colony was picked up with the help of an inoculating needle and cross-streaked on another NA plate for purification. The procedure was repeated until all the colonies developed on the media surface resembled to that of the first selected colony. A single well-separated colony was transferred to NA slant and assigned with an isolated number. To assess the pathogenicity of bacterial isolate, fresh, apparently healthy mushroom buds at the egg stage was collected from mushroom growers and placed them in new polythene bags and brought to the laboratory Pathogenicity test was conducted as described by Kumar et al., (2018). The pure bacteria colonies obtained from the colonies growing on the nutrient agar were later identified based on morphological and biochemical tests as reported by CMI (2010). DNA extraction and BLAST analysis of 16S rRNA sequence homology was conducted as described by Joko et al., (2014). This sequence was submitted to the GeneBank database under accession number OQ410475. Water-based crude extracts were collected from fourteen plant species (Table 1) chosen for their common use in Ayurvedic medicine. Plant extracts on the growth of test bacteria were conducted according to Gonelimali et al., (2018).
 

 

 
Twelve different antibiotic discs which were commercially available (Hi –media) were tested against the test bacteria. Bacterial suspension was transferred and spread on NA plates following the same procedure as described for testing of plant extracts. A set of three numbers of antibiotic discs were placed on the previously inoculated plates. The inoculated plates with antibiotic disc were incubated at 27±1°C for 24-48 h. Zone of inhibitions from the center of the antibiotic disc were measured in millimeter. (mm).

Bacteria associated with Volvariella mushroom
 
One bacterial isolate was detected exibiting browning of the bulbous base of the Volvariella sp. was the major diagnostic symptom.The symptoms were exhibited in diseasesd mushrooms and not present in healthy mushrooms which looks white from base to stipe. The SEM of the bacteria was taken as described earlier which revealed that the associated bacteria was rod-shaped and capsulated measuring 1.33-1.69 µm (Fig 2). Browning of the bulbous base linked to the vulva and fungal mycelium suggests that multiple pathogens association in paddy straw mushroom diseases including a novel symptom, complete rotting was found associated with one isolate. Systematic studies are required for better conclusive results. Similar type of symptom were not earlier reported and hence it was the first report of this paddy straw mushroom disease.
 

 
Identification of bacterial isolates based on morphology, utilization of carbohydrates, biochemical characterization  and molecular test
 
Morphological characterization revealed bacterial isolate was rod-shaped, capsulated, measuring 1.33-1.69 µm. No flagella could be detected during gram staining technique. The colonies on nutrient agar appeared as circular, convex, glistering and pink to red in color. Production of acid from carbohydrate was also revealed that the bacterium was found positive in reaction against xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melobiose, sucrose, L-arabinose, manose, rhamnose, cellobiose and sorbose. Results of the biochemical test revealed that the isolate showed positive reaction with regard to esculin hydrolysis, citrate, malonate, lysine and orthinine utilization; urease production, nitrate reduction, H2S production and casein hydrolysis. The growth of bacteria at 27°C was positive and at 47°C red coloration disappeared and producing light yellow colored colony (Table 2 and 3). The amplification of the 16S rRNA of this bacterial strain (accession number OQ410475) was generated. A similarity search of the 16S rRNA gene sequence against the NCBI GeneBank database showed a 99 to 100% homology to known sequences of Serratia marcescens. A phylogenetic tree (Fig 3) was constructed using sequences in databases corresponding to strains of Serratia group which one you have followed in which Serratia marcescens was found and the isolate was identified as Serratia marcescens. In the present study, Serratia marcescens caused browning of bulbus base followed by rotting of sporophore of market samples of Volvariella sp. In India Kannaiyan, (1978) reported Pseudomonas sp. bacterial rot disease of Volvariella sp.  Because pseudomonas tolasii due to its widespread distribution, was mainly studied pathogen. It affects a variety of mushroom hosts including A. bisporus, A. bitorquis, Pleurotus ostreatus and Pleurotus eryngii causing significant losses to mushroom cultivation Bradbury, (1987); Soler-Rivas et al., (1999). Munsch et al., (2000) was also reported the association of Pseudomonas costantinii with formation of brown blotch disease of Agaricus bisporus. The present revealed the association of Serratia marcescens which was responsible for causing the browning and rotting of commercially cultivated paddy straw mushrooms (Volvariella sp.) was found to be the first report in india.
 

 

 

 
Pathogenic ability of Serratia marcescens
 
Serratia marcescens induced 100% rot in 72 hours, while the healthy (Mushrooms) control remained unaffected. Upon reisolate matched with the original bacterial culture.
 
Sensitivity of Serratia marcescens to different plant extracts
 
Crude plant extracts with water base were collected from fourteen plant species and tested against the Serratia marcescens. Zone of inhibition of bacterial growth was recorded and presented (Table 4) and (Fig 4). Trigonella foenum and Aloe barbadensis plant extracts were found most effective in inhibiting the growth of the bacterium. Whereas, Psidium guajava, Elettaria cardamomum, Curcuma longa and Tridax procumbens extract could not inhibit the growth of the test bacterial species. The rest of the plant extracts differed in their effectiveness against Serratia marcescens. Since plant extracts recorded potent antibacterial activity against harmful bacteria are considered to be acceptable as eco-friendly alternatives. As paddy straw mushroom is produced in 15 days, the use of chemicals were banned for use in mushroom beds. Paddy straw mushroom grown by using paddy straw as substrate. Therefore, mixing plant parts or their extracts along with the substrate having antibacterial properties was found better proposition for the management of bacterial diseases as they inhibit the bacterial growth to establish itself within the mushroom beds.
 

 

       
In the present study, sensitivity of Serratia marcescens to fourteen plant extracts were studied out of which the bacterium showed sensitivity to ten plant extracts. Todorović et al., (2016) tested essential oil of twenty plants extracts Pseudomonas tolaasii, the causal agent of a bacterial brown blotch disease of cultivated mushrooms and observed that P.  tolaasii was most sensitive to peppermint oil. Trigonella foenum plant extract inhibited the growth of the test bacterial pathogens of paddy straw mushrooms because Trigonellin is the essential oil present in Trigonella foenum. Confirmative studies are required with regard to the temperature sensitiveness of the active ingredients and toxicity against the mycelial growth of mushroom fungus before giving final recommendation for use.
 
Sensitivity of the Serratia marcescens to different antibiotics
 
Commercially available antibiotic discs were tested against the bacterial species by recording growth inhibition zone formed on inoculated media plates. The results presented in (Table 5 and Fig 5) revealed that among different antibiotics Cefaclor recorded maximum zone of inhibition followed by Ciprofloxacin. Two test antibiotics could not inhibit the growth of bacterial species. Among the twelve antibiotics tested, the bacterium was found sensitive to nine antibiotics. The highest inhibition zone of 65.6 mm of bacterial growth of Serratia marcescens was recorded in case of Cefaclor whaere as Clarithromycin recorded the lowest zone of inhibition.
 

 

       
Worldwide survey has shown that representative of Bacterial illnesses infects every major genus of edible mushrooms including Agaricus, Pleurotus, Lentinus, Flammulina, Volvariella and Auricularia. As more intense farming techniques are adopted, pseudomonadales are the primary cause of most mushroom crop losses. Manipulation of the environment in the growing room and application of regular drenching of chlorinated water to casing layer has been suggested by Fermor, (1986). Application of terramycin 9 mg per square feet, streptomycin (200 ppm), oxytetracycline (300 ppm), kasugamycin and kanamycin were found effective in managing the bacterial diseases of mushrooms as reported by Sharma et al., (2007). All the above recommendations have been tried in management of bacterial diseases of commercially grown mushrooms other than Volvariella sp. The cultivation methods for Volvariella sp. seems to create an environment suitable for the growth and establishment of various bacterial pathogens. In the present study Cefaclor and Ciprofloxacin antibiotics were found effective against the bacterial species. Detection of bacterial pathogens present in the mushroom bed and application of selected antibiotics is thought to be a difficult proposition in managing bacterial diseases of Volvariella sp. Therefore, more investigations are required for selection of a broad-spectrum antibiotic.

The bacterial pathogen was isolated from Volvariella Mushroom exhibited bacterial rot symptom. The bacterial isolates were studied for their pathogenic ability in causing rot of apparently healthy mushroom buds. Several morphological and biochemical characteristics of bacterial isolate were studied and identified as Serratia marcescens and the bacterium was studied for its sensitivity to different plant extracts and antibiotics. Study of the sensitivity of bacterium against Fourteen different plant extracts revealed that it was more sensitive to plant extract of Trigonella foenum and Aloe barbadensis whereas the plant extracts of Psidium guajava, Tridax procumbens, Curcuma longa with Elettaria cardamomum were found to be ineffective in inhibiting the growth of bacterium. Among 12 antibiotics, cefaclor was found to be most effective in inhibiting the growth of the bacterium whereas ampicillin and Cefoperazone were found to be ineffective. The rest of the antibiotics differed in their sensitivity to the bacterium. More studies required to determine the suitable measures combining sanitation, integrated management of bacterial diseases in addition to use of plant extracts and antibiotics.

Authors do not have any conflict of interest to declare.