White mold on pea caused by Sclerotinia sclerotiorum : a new threat for pea cultivation in Bangladesh

A new disease causing the tan to light brown blighted stems and pods has occurred in 2.6% pea (Pisum sativum L.) plants with an average disease severity rating of 3.7 in Chapainawabganj district, Bangladesh. A fungus with white appressed mycelia and large sclerotia was consistently isolated from symptomatic tissues. The fungus formed funnel-shaped apothecia with sac-like ascus and endogenously formed ascospores. Healthy pea plants inoculated with the fungus produced typical white mold symptoms. The internal transcribed spacer sequences of the fungus were 100% similar to that recovered from an epitype of Sclerotinia sclerotiorum, considering the fungus to be the causative agent of white mold. Mycelial growth and sclerotial development of S. sclerotiorum were favored at 20°C and pH 5.0. Glucose was the best carbon sources to support hyphal growth and sclerotia formation. Bavistin and Amistar Top inhibited the radial growth of the fungus completely at the lowest concentration. In planta, foliar application of Amistar Top showed the considerable potential to control the disease at 1.0% concentration until 7 days after spraying, while Bavistin prevented infection significantly until 15 days after spraying. A large majority (70.93%) of genotypes including tested released pea cultivars were susceptible, while six genotypes (6.98%) appeared resistant to the disease. These results could be important for management strategies aiming to control the incidence of S. Sclerotinia and eliminate yield loss in pea.


Introduction
Pea (Pisum sativum L.) is an herbaceous annual plant in the family Fabaceae or Leguminosae. It is an important and popular food crop grown in many countries all over the world, especially for its edible seeds. Globally pea is the fourth leading legume in terms of consumption with a total production of 10.2 million tons [1]. Pea is appreciated primarily for its high nutritional value with 23 to 25% protein [2]. 4 sclerotia within mycelium and in cavities of infected plant parts. Sclerotia is a vital part of the disease cycles. Sclerotia can germinate vegetatively and produce mycelium for infection, or they most often carpogenically form apothecia, a sexual fruiting body produced after a period of dormancy. Within the apothecia, the tubular asci develop which finally discharge ascospores into the air. The airborne ascospores initiate infection on host foliage. Temperature and pH value of substrates have a strong influence on mycelial growth, cellular morphology, metabolite biosynthesis, and sclerotial formation of the fungus [18]. Equally, nitrogen and carbon resonate mycelial growth and sclerotial formation of the fungus and are considered as initial nutrient sources for infection, [19,10]. Knowledge of the environmental conditions conducive to S. sclerotiorum inoculum formation is essential for the successful management of white mold disease.
The disease needs to be satisfactorily controlled to reduce yield losses. Attaining good control of white mold is a challenging task in all crops. Disease management requires the application of a wide range of strategies. In practice, fungicide application is the core strategy to protect crop plants from white mold [20]. Similarly, host resistance is considered the safest and most economical way of controlling the disease. Since the white mold of pea is new in Bangladesh, information about pathogen characteristics, effective fungicides, and resistance sources need to be clarified for successful management of the disease. The present study was designed to isolate and identify S. sclerotiorum causing white mold in pea, determine conditions conducive to optimum growth and sclerotia formation, decide effective fungicides for controlling the pathogen, and explore effective sources of resistance.

Disease survey in the field and sample collection
A survey was conducted for pea diseases in farmers' fields in Chapainawabganj district, Bangladesh from 21 to 23 January 2018. A total of 15 farmers' fields were randomly selected. Pea fields were at 5 the late growth stage when plants were bearing pods but had not senesced. In each field, five 1.5 m × 1.5 m quadrats located at each of four corners and the center were used for visual examination of disease.
Disease incidence and severity were recorded simultaneously in each of the fields. Disease incidence was assessed as the percentage of symptomatic plants compared to total plants. Severity was calculated in the field by adopting a scale of 0-5, where 0 = no symptoms, 1 = up to 10%, 2 = 11-35%, 3 =36-65%, 4 = 66-90%, and 5 = 91-100% of the foliage affected by the pathogen [21]. Diseased stems and pods were collected from each of the infected fields and returned to the laboratory for examination.
Sclerotia were separated from infected plant parts and assessed for their morphological characteristics.

Fungal isolation and preservation
Three infected plants with blighted stems and pods with sclerotia and visible mycelium were arbitrarily taken for fungal isolation. Two infected plant parts (one stem section and one pod) and four sclerotia were sampled from each of the three plants. Infected plant parts were cut into 1 cm pieces, while sclerotia were divided in half. The cut pieces were sterilized in 75% ethanol for 1 min, washed three times in sterile distilled water, and dried between two layers of blotter paper. Cut off sclerotia and infected plant pieces were aseptically plated onto acidified potato dextrose agar (PDA) medium amended with 1 ml of 85% lactic acid in 9-cm-diameter Petri plates. Similarly, two fragments of mycelium were removed from the surface of infected stems and pods of each of the three plants with forceps and placed directly on PDA. All plates with infected plant parts, sclerotia, and mycelium fragments were incubated at 25±1 O C in the dark for 5 days. Emerging mycelial colonies were transferred to new PDA plates and pure isolates were obtained by hyphal tip isolation. Pure isolates were cultured on PDA slants and preserved at 4°C.
Morphological characterization of the isolate 6 Each of the isolates was transferred on PDA in 9 cm diameter Petri dishes and cultured at 25 O C in the dark for a week. Colony characteristics of the fungus were studied with the naked eye. Permanent slides were prepared from the colony and hyphal characteristics were examined under a light microscope at different magnification. Sclerotia produced on the medium were examined for morphological features.

Production of apothecia from sclerotia
Sclerotia were collected from each culture plate and after surface sterilization, 50 sclerotia were placed in five 9 cm-Petri dishes filled with wet sterile sand and incubated at 4ºC for 6 weeks. The Petri dishes having germinating sclerotia were transferred to another incubator at 20ºC under scattered fluorescent irradiation (260 μmol/m 2 /s) until apothecial discs were developed [22]. Fifteen apothecia and the ascospores produced from sclerotia were examined under the microscope (40× and 100×) for morphological features.

Pathogenicity test of the isolates
A pathogenicity test was conducted to satisfy Koch's postulates, and it was repeated twice. The main cultivation varieties of BARI Motorshuti 3 were used for testing. Three pure isolates, one each obtained from infected tissues, sclerotia, and mycelial fragment, were selected for the pathogenicity test. Seeds of pea variety 'BARI Motorshuti 3' were surface sterilized and sown in earthen pots filled with sterile soil in the net house. In each of two trials, five plants were inoculated with each isolate and the same numbers were included as a control. When the seedlings were four-week-old (five to seven fully 7 expanded leaves), 5 mm-diameter plugs from actively growing margins of fungal colonies were placed on superficially wounded stem approximately 10 cm above the soil line. For five control plants, only sterile PDA plugs were used. All plugs were sealed with moistened cheesecloth. The inoculated plants were placed in a dark humid chamber for 48 h at 22±1°C. Then the inoculated seedlings were shifted to a net house and grown under observation for 20 days. Pathogenicity of the isolates was also tested on pea pods and leaves by placing a mycelial plug of the fungi onto an incised pod and leaf surface in a similar manner. The suspected pathogen was re-isolated from inoculated tissues that exhibited symptoms of white mold. It was determined whether the original and the re-isolated strains were identical.

Molecular identification of S. sclerotiorum isolate PSP-1
Total genomic DNA was extracted from the mycelia of S. sclerotiorum isolate PSP-1 as described by Toda et al. [23]. Polymerase chain reaction (PCR) was conducted with forward primer ITS-1 (5'-TCC GTA GGT GAACCT GCG G-3') and reverse primer ITS-4 (5'-TCC TCC GCT TAT TGATAT GC-3') [24] to amplify rDNA-ITS regions of the fungal isolates. PCR was done following the method described by Hayakawa et al. [25]. Two complete sequences were obtained for each ITS region and the BLAST search program was used to search for nucleotide sequence homology in GenBank. Highly homologous sequences were aligned using Clustal-X version 2.0.11 and manually adjusted as required. Neighborjoining trees were generated using MEGA X version. Bootstrap replication (1000 replications) was used as statistical support for the nodes in the phylogenetic trees. Based on maximum sequence homology percentage, query coverage, and the lowest E value, ITS sequence data of four isolates of S. sclerotiorum were selected for phylogenetic analysis. Additionally, three isolates of each of S. minor and S. trifoliorum were included in this analysis, while Hypocrea lixii was used as an outgroup taxon (GenBank accession no. FJ 861393.1).

Effect of temperature on mycelial growth and sclerotia formation of S. sclerotiorum isolate PSP-1
The effect of temperature on mycelial growth of S. sclerotiorum was determined by growing the fungus on PDA at different temperature regimes 5, 10, 15, 20, 25, and 30°C in two independent trials. In each trial, PDA plates (9-cm-diameter) were prepared and inoculated at the center with a 5 mm-diameter mycelium plug excised from a 6-day old S. sclerotiorum culture. There were three replicates for each temperature regime and each replicate consisted of three Petri plates. Inoculated plates were incubated at each of 5, 10, 15, 20, 25, and 30°C in the dark. The radius of colonies was measured at 24, 48, and 72 h after incubation. Cultures were continued for 15 days for maximum sclerotial yield. The number, diameter, and weight of sclerotia were taken.

Effect of pH on mycelial growth and sclerotia formation of S. sclerotiorum isolate PSP-1
The growth and sclerotia formation of S. sclerotiorum was examined at a diverse pH range. PDA media were prepared in 9-cm-diameter PDA plates and adjusted their pH to 3.0, 5.0, 7.0, and 9.0 using 0.1 N HCl and NaOH. There were three replicates for each pH range and each replicate consisted of three Petri plates. Petri plates were inoculated at the center with a 5-mm-diameter mycelium plug excised from a 6-day old S. sclerotiorum culture. The fungal cultures were incubated at 20°C in dark. The radius of colonies was measured at 24, 48, and 72 h after incubation. Cultures were monitored for sclerotial development and then the number, diameter and weight of sclerotia were taken after 15 days. The experiment was repeated twice.

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Effect of carbon sources and pH on mycelial growth and sclerotial formation of S. sclerotiorum isolate

PSP-1
A minimal medium composed of peptone at 4.0 g/ l, carbon sources (sucrose, mannitol, glucose, fructose or soluble starch) at 20 g/l, MgSO4 at 0.5 g/l, KH2SO4 at 1.0 g/l, and agar at 20 g/l was used.
The pH of the medium was adjusted to 4.0, 5.0, 7.0, and 9.0 using 0.1 N HCl or NaOH before autoclaving. There were three replicates for each pH and three 9-cm-diameter Petri plates were prepared for each replicates Mycelium plugs of 5-mm-diameter were cut from the growing edge of a 6-day S. sclerotiorum culture and placed at the center of each plate. The cultures were then incubated at 20°C in dark. The mycelial growth of the fungus was determined by measuring colony diameters at 72 h after incubation. The same cultures were continued for 15 days to induce maximum sclerotial formation.
Sclerotial number and weight were taken. The experiment was repeated twice. Fungicides were added at a concentration of 10, 50, and 100 ppm in an autoclaved PDA medium following poisoned-food techniques [26]. The pH was adjusted to 5.0 using 0.1 N HCl. Agar disk of 5 mm diameter of fungal culture was excised from a 3-day-old culture and placed in the center of Petri plates having different fungicidal concentrations. There were three replicates of each treatment and each Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 August 2020 doi:10.20944/preprints202008.0570.v1 replicate consisted of three plates. The plates without fungicides worked as control. The inoculated plates were incubated at 20°C. After 7 days of incubation when the fungus in control plates covered the whole plate, the diameter of the radial growth was recorded. The percent inhibition (PI) of the fungus over the control was calculated using the formula of Sundar et al. [27].

Design of Experiments and Analysis of Data
Statistix 10 (Analytical Software, FL, USA) was used for statistical analysis. The experimental design was completely randomized, consisting of at least 3 replications for each treatment. The experiment was repeated at least twice and treatments were compared via ANOVA using the least significant difference test (LSD) at 5% (P ≤ 0.05) probability level. Data were transformed as and when necessary using the arcsine transformation method.

Detection of the disease in the field
In our survey, pea plants with tan to light brown blighted stems and pods were visually detected in all the surveyed fields (100%) in Chapainawabganj district, Bangladesh. The disease incidence accounted for 2.6% infected plants with an average disease severity rating of 3.70. Infected plants were at the late growth stage, but had not senesced. Some of the infected plants died prematurely and showed a typical wilting symptom. The most conspicuous symptom was the occurrence of the necrotic pod surface covered by a thick white fuzzy mycelial growth with large black sclerotia (Fig. 1A). When splitting the infected stem longitudinally, white mycelial growth with sclerotia was also detected along the stem pith. The sclerotia were round to irregular in shape and measured 3.5-14.8 × 1.9-5.3 mm in size. 13 All mycelial colonies from sclerotia, mycelium fragments, and symptomatic tissues yielded identical cultures on PDA. A white closely appressed and thin mycelial growth radiated over the PDA plate, but it was relatively thick at the colony margin (Fig. 1B). The reverse colony showed a salmon buff color.

Fungal isolation and characterization of the pathogen
The hyphae were aseptate, hyaline, branched and multinucleate (Fig. 1C). No conidium or conidiophore was produced. However, sclerotia were produced in all cultures after 10 days. Sclerotia were commonly formed in-ring and usually close to the periphery of the colony. Three distinct phases of sclerotial development were observed. Whitish aggregates of mycelia appeared (initiation stage) after 5 days, turned beige-colored after 7 days (development), and developed into the dark (maturation) after 10 days of growing ( Fig. 1D-F). The number and weight of sclerotia produced per PDA plate ranged from 18 to 31, and 7.38 to 15.81 g, respectively. Sclerotia were of different shapes and sizes with a black outer rind and a white inner cortex. Individual sclerotium measured up to 7 mm long and 4 mm wide.

Production of apothecia, asci, and ascospores
Apothecia were produced in 5 to 6 weeks of incubation. One to four apothecia arose from a single sclerotium. Apothecia were tan to beige. Receptacles were flared, somewhat concave when immature, but funnel-shaped at full maturity. Young apothecia were 1 to 2 mm in diameter, while mature apothecia were 4 to 7 mm in diameter with a stipe length of 7 to 15 mm. Asci from apothecia were cylindrical with a tapered base. Each ascus had eight ascospores (Fig. 1G). Ascospores were uniseriate, hyaline, one-celled, smooth, ellipsoid, and uniform in size (Fig. 1H-I). Paraphyses were abundant in the hymenium.

Pathogenicity tests
In

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The mycelial growth of S. sclerotiorum was affected by the variation of temperature ranging from 5°C to 30°C. At 5°C and 10°C, the growth of the isolate was very sparse as it was visible 72 h after incubation. When the temperature increased to 15°C, the growth occurred faster (Fig. 3A) and increasing temperature further to 20°C resulted in maximum mycelial growth of the fungus. As the temperature increased to 25°C, the growth declined more than three folds 72 ha after incubation. Further increasing temperature to 30°C arrested mycelial growth completely and no redial growth was observed 72 ha after incubation (Fig. 3A). There was also a significant effect of temperature on sclerotia  (Fig. 3C).

Effect pH on mycelial growth and sclerotium formation of S. sclerotiorum isolate PSP-1
The pH of the culture media had a significant effect on mycelial growth of S. sclerotiorum. Generally, the fungus was able to grow over a wide pH range, with the highest growth observed at pH 5.0 (Fig.   3B). When the pH was lesser or greater than 5.0, the mycelial growth of the pathogen decreased.
Accordingly, the lowest mycelial growth of the pathogen was observed at pH 3.0 followed by pH 9.0 and 7.0. Sclerotia formation has also been shown to be affected by pH conditions. Although sclerotia were formed at diverse pH ranges between 3.0 and 9.0, the highest sclerotial yield resulting from the higher sclerotial number and greater individual sclerotial weight was recorded at pH 5.0 followed by pH 3.0. Oppositely, the lowest sclerotial yield resulted from the fewer sclerotial numbers and lower individual sclerotial weight was observed at pH 9.0 followed by pH 7.0 (Fig. 3D). This suggests that the sclerotium formation of S. sclerotiorum is favored under acidic than under neutral and alkaline conditions.

PSP-1
In all carbon source media, the fungus grew and produced sclerotia under acidic, neutral, and alkaline conditions. However, good growth and sclerotial yield were achieved under acidic than neutral or alkaline conditions. Precisely, the highest mycelial growth rate and sclerotial yield were attained at pH 5.0 regardless of which carbon source was used. Decreasing pH to 4.0 or increasing pH to 7.0 or 9.0 resulted in progressively slowed mycelial growth and lower sclerotial yield. Accordingly, the lowest mycelial growth and sclerotial yield were observed at pH 9.0. Among the carbon source media, glucose and mannitol favored the maximum mycelial growth, while starch supported the least. Similarly, the highest sclerotial yield in terms of the number and weight of sclerotia was observed in glucose followed sucrose, whereas the lowest was recorded in starch ( Table 2).

In vitro efficacy of fungicides on the growth of S. sclerotiorum isolate PSP-1
Six fungicides were tested at three concentrations (100, 50, and 10 ppm) for their ability to inhibit mycelial growth of S. sclerotiorum. Among the tested fungicides, Bavistin and Amistar Top were the most effective in inhibiting the radial growth. Mycelial growth was completely inhibited by them at all three concentrations ( However, Bavistin was able to restrict the disease significantly and the control efficacy was 80%.

Screening of pea genotypes against S. sclerotiorum isolate PSP-1
Visible water soaking and leaf necrosis symptoms appeared under the plug after 48 h. By 96 h, the water soaking and necrotic regions reached the leaf margin in some leaves with green leaves becoming necrotic and discoloring to a brown. No such symptoms were observed in the non-inoculated control leaves. A significant difference in disease response was observed across tested cultivars (Fig. 4). Among  (Table 5).

Discussion
Pea is an important crop and widely used for feed and food. A new disease with a significant yield loss was detected in the pea field in Chapainawabganj district, Bangladesh since 2018. The suspected disease showed a range of characteristic symptoms that are distinctive from other pea diseases such as downy mildew or Ascochyta blight [29]. Proper characterization of the causal pathogen and subsequent identification of effective fungicides and resistant sources are important to gain a clearer understanding of the disease and to minimize future losses. This study represents the first attempt to characterize a new disease of pea according to their morphological traits, pathogenicity tests, and phylogenetic analyses of ITS regions.
Based upon the observed field symptoms of the disease, it was presumed that the new disease would be white mold caused by S. sclerotiorum [30]. The finding that the isolated fungus has cultural and morphological characteristics similar to those of S. sclerotiorum e.g., a white appressed mycelial growth, the formation of a ring of sclerotia on the plate, lack of conidia and conidiophore, existence of multinucleated hyphae, and production of apothecia, asci, and ascospores provided further evidence for white mold disease [31,32]. Moreover, the induction of white mold symptoms in pea, indistinguishable from those observed in the field, by the isolated fungus fulfilled Koch's postulates and confirmed the pathogenicity establishment of S. sclerotiorum. Finally, sequence comparisons and phylogenetic analyses showed that the fungus was most closely related to S. sclerotiorum. Thus, these results established that the disease under inspection on pea is white mold caused by a fungus S. sclerotiorum.
Although S. sclerotiorum has been reported to be the pathogen of the white mold of a pea in other countries [33,34], this is the first documented occurrence to our knowledge of a strain infecting pea in Bangladesh [9]. In Bangladesh, S. sclerotiorum was previously reported on hyacinth bean, jackfruit, and okra [10,17,28,34], implying that the pathogen has the potential to expanse to other host species.
The wide host range of the pathogen poses an epidemiological threat and makes the management of the disease very challenging.
The white mold pathogen S. sclerotiorum is sensitive to temperature. Temperature influences the growth and development of the fungus because of its effect on the enzymes involved. Although S.
sclerotinia can grow under a wide range of temperatures, the optimal temperature for mycelial growth is 20°C. The radial growth rate of the mycelium decreased at temperatures below 10 O C and above 25°C.
Similarly, the formation of sclerotia was at a temperature between 15 to 25 O C, with an obvious maximum at 20°C. These findings were in agreement that S. sclerotiorum has a preference for growth in subtropical climates [28,32]. The 20 O C optimum reported for growth coincides with the temperatures reported as favoring infection and pathogenesis by S. sclerotiorum [36]. Knowledge about environmental factors affecting the pathogen could contribute to the development of new methods of managing the disease. Therefore, early sowing might be effective at controlling white mold in pea.

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The external pH puts a significant influence on fungal morphogenesis through a highly complex mechanism [37]. Moreover, pH has been involved as a major regulatory factor for procedures linked to development, pathogenicity, and virulence of S. sclerotiorum [18]. It is known that pH may act in several different ways, such as influencing enzyme action, altering metal solubility, modifying surface reactions, and preventing or facilitating the entry of vitamins, organic acids, and minerals into the hypha [38]. In general, fungi tolerate relatively narrow ranges of pH. However, mycelial growth and sclerotium formation of the S. Sclerotiorum isolate in this study were found to occur over a broad pH range, from pH 3 to 9, suggesting that S. Sclerotiorum isolate pathogenic to pea is likely to proliferate in most agricultural soils in which peas are grown. Yet, the optimal growth and sclerotium formation of the fungus was observed at a slightly acidic pH value of 5.0, indicating that S. sclerotiorum isolate exhibited the best growth characteristic of a fungus specific for acidic habitats. Since pH is not a unitary actor, the reasons for the preference for slightly acidic conditions for growth and sclerotial development of the fungus are unclear and warrants further investigation. Several hypotheses have been postulated concerning the effect of pH on mycelial growth and sclerotia production. Membrane permeability is affected by pH, therefore the ability of the fungus to take up nutrients required for mycelial growth and sclerotial production may have been affected at different media pH [39]. Oxalic acid production by S. sclerotiorum is also governed by pH [40]. The fungus may produce substantially greater amounts of oxalic acid and support mycelial growth and sclerotial formation as long as the pH of the culture is 5.0 [28].
Carbon assimilation is necessary for pathogenic fungi to survive, grow, and persist within a host. The carbon metabolism of S. sclerotiorum is strongly influenced by the nature of the carbon compound. Carbon compounds perform two important functions in the metabolism of fungi. They supply the carbon needed for the synthesis of cell components and form the primary source of energy through their oxidation [41]. There are no available accounts of the carbon requirements of S.
sclerotiorum isolate infecting pea. In this study, S. sclerotiorum isolate metabolized a broad range of sugars. This indicates the efficient assimilation of different carbon sources by the fungus, hence enhancing the fitness of this pathogen in the host. Of the six-carbon sugars, glucose appears to be the best carbon source that supported the highest mycelia growth and sclerotium formation at pH 5.0.
Glucose is a biologically most important carbon compound that is utilized for growth and energy by virtually all cultivated fungi [42]. Good growth and sclerotium formation were also observed with mannitol and sucrose. Simple carbohydrates (glucose, mannitol, and sucrose) are utilized by S.
Although many fungicides show in vitro fungitoxic activity, a few of them prove effective against pathogens in plants [50].