Emergence of Choanephora Blight in Okra in Eastern India, Disease Severity, Etiology, and Fungicidal Management

doi:10.21203/rs.3.rs-4952640/v1

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Emergence of Choanephora Blight in Okra in Eastern India, Disease Severity, Etiology, and Fungicidal Management

https://doi.org/10.21203/rs.3.rs-4952640/v1

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Okra is an important vegetable crop extensively cultivated in India. During the months of July-October 2019 occurrence of Choanephora blossom blight has been documented first time at the research farm of IIHR-Central Horticultural Experimental Station, Bhubaneswar, as well as from farmers' fields and research plots of Odisha University of Agriculture and Technology, Bhubaneswar, Odisha located in Eastern India. Subsequent survey to six major okra growing districts of Odisha revealed the incidence of up to 35.0 percent. Hence efforts were made to study the detailed symptomatology, and etiological factors. Choanephora cucrbitarum was identified as the causal agent based on morphological characteristics, molecular characterization as well as pathogenicity assay. In-vitro bio-efficacy of fungicides demonstrated the effectiveness of triazole group of fungicide against the test pathogen offering promising prospects for disease management in okra cultivation.

Blossom blight

Choanephora cucurbitarum

leaf blight

okra

pod rot

Okra, also known as lady's finger (Abelmoschus esculentus L.), occupies an important position as a vital vegetable crop in India. Its significance transcends borders, finding cultivation across tropical and subtropical regions worldwide, including India, Japan, Turkey, Iran, southern United States (Qhureshi, 2007). India, being a leading global producer of okra contributes around 67.1% of the total okra output, boasting an expansive cultivation area of 509.02 million hectares with a remarkable yield of 6094.94 million metric tonnes. This narrative extends to Odisha, located in Eastern Part of India, where cultivation spans 64.07 million hectares, with yield of 566.88 million metric tonnes (Horticultural statistics at a glance, 2018). Okra's versatility is striking; it serves as both a culinary delight as well as a repository of nutrition. Notably, okra’s nutritional profile boasts vitamin C and carbohydrates. With high fiber content, okra's potential extends to managing blood sugar levels, making it as a beneficial component for diabetes management (Ngoc et al., 2008).

However, the okra landscape is not without its challenges. Evolving farming practices, variable weather patterns, and the introduction of novel germplasm are catalysts for numerous okra diseases, which were not reported in India. Damping off (Pythium sp. and Rhizoctonia sp.), Fusarium wilt, Powdery mildew (Erysiphe cichoracearum), Cercospora leaf spot (Cercospora abelmoschi), Yellow Vein Mosaic Virus (YVMV), Alternaria leaf spot, Okra leaf curl, Enation leaf curl and Choanephora blight (Choanephora cucurbitarum) cause considerable crop loss world-wide (Raid and Palmateer, 2006; Ati and Tohany, 2004). Under our Eastern India condition Choanephora blight has emerged as a significant concern, in recent years on many crops (Ganesan et al., 2021; Das et al., 2017). Occurrence of this disease is reported from Korea, Egypt, China, Malaysia, and India (Park et al., 2015; Hussein and Ziedan, 2013; Liu et al., 2019; Siddiqui, 2006; Naseema and Wilson, 1993).

A kind of wet rot symptom, quickly covered by a lush array of shimmering silvery conidiophores, devoid of external vegetative mycelium, a distinctive character of Choanephora was observed as early as in 1920 by Dastur. Adebanjo and Dede (1985) identified that this disease led to water-soaked fruits, often starting at tips, causing growth stagnation, premature fruit abscission, or shrinking. Pathogen prevalence notably delayed flowering, increase rot rates, and subsequently led to decreased yield in Okra (Balogun and Babatola, 1999). Various group of researchers identified a fungus which infect flowers, immature pods, and leaves during different stages of okra crop development, resulting in symptoms of wet rot, pod blight, and leaf blight (Liu et al., 2019; Hussein and Ziedan, 2013; Park et al., 2015).

Presently only two species have been recognised within the genus Choanephora such as C. infundibulifera and C. cucurbitarum (Kirk, 1984). According to Kirk's taxonomic criteria, the shape of the sporangiolum and the existence of striations determine their differentiation. Both species frequently infect a wide array of hosts - angiosperms (monocotyledons and dicotyledons) and gymnosperms, inducing leaf blights and fruit rots (Farr and Rossman, 2014), however, C. cucurbitarum has been acknowledged as a more extensive plant pathogen than C. infundibulifera, affecting diverse hosts (Park et al., 2016).

The fungus infects chilli, Amaranthus sp., Dolichos bean, and yard long bean with a range of symptoms, including leaf blight, shoot apex blight, stem soft rot, dieback, and pod decay mainly in coastal belts of Odisha, West Bengal and Kerala (Ganesan et al., 2021; Das et. al., 2018 and Kurian et al., 2018).

The fungi initially displayed non-parasitic tendencies however, a significant alteration in weather patterns, marked by higher relative humidity (RH), wetness, and temperature, specifically at 25°C and 70–90% RH, led to its transition into a facultative parasite (Dastur, 1920). Favourable conditions during the monsoon season, characterized by warm and humid weather, particularly prompted more pronounced infections (Hyde et al., 2014; Das et al., 2018; Kwon and Hyeong, 2005; Kwon et al., 2001; Kurian et al., 2018). However, no control measures have been identified to manage the disease either in India or from other countries.

Due to the escalating damage and substantial yield loss in okra cultivation regions of Odisha, India, coupled with the pathogen's broad host range, the present research aimed to study the severity of Choanephora blight across the state of Odisha, its etiology and management.

Survey, isolation and purification of the fungi

During the kharif season of 2019-20, a survey was conducted in thirty villages across Odisha's major okra-growing districts, including Jajpur, Cuttack, Khordha, Bhadrak, Balasore, and Mayurbhanj, to record the prevalence and disease incidence in okra. The number of plants infected with Choanephora rot was counted, and the Percent Disease Incidence (PDI) was computed using the following formula:

$$\:PDI=\frac{Number\:of\:infected\:plants\:}{Total\:number\:of\:plants\:observed}\times\:100$$

Infected leaf samples were first separated from dirt particles using tap water and then chopped into 3–5 mm pieces using a sterile knife to start the isolation procedure. The leaf pieces were surface-sterilized for one minute in a 2% sodium hypochlorite (NaOCl) solution, and then they were thoroughly cleaned three times using sterile distilled water. After that, the samples were put on potato dextrose agar (PDA) with 0.1 mg mL − 1 streptomycin sulphate and left to incubate for three to five days at 28 ± 2°C. Periodically, the plates were examined to look for signs of fungal development. Hyphal tips growing from the pieces of the leaf samples were transferred onto fresh PDA plates to obtain pure culture of the fungus.

Symptomatology

The symptoms on different parts of the plant and sporulation of the fungus were studied and documented. All the standard meteorological parameters like temperature, relative humidity, rainfall and sunshine hours during the crop period was obtained from the Meteorological Observatory, Department of Agrometeorology, OUAT, Bhubaneswar. The severity of the disease was graded on a 0–5 scale, with 0 indicating no infection, 1 indicating 1–19 percent infection, 2 indicating 20–39% infection, 3 indicating 40–59% infection, 4 indicating 60–79% infection, and 5 indicating 80–100% infection. For the purposes of the epidemiological study, disease severity scores were transformed to a Percentage disease severity (PDS) (Wheeler, 1969).

$$\:PDS=\frac{Sum\:of\:numerical\:ratings}{Number\:of\:plants\:scored\times\:Maximum\:score\:on\:scale}\times\:100$$

Morphological and cultural characterization

For morphological assessment, the pathogen was cultured on PDA medium in 90 mm Petri plates at 27 ± 1℃. Within 24–48 hours of inoculation, mycelial development reached the plate's edge. To determine the optimal medium for growth and sporulation, the isolated fungal pathogen was cultivated in seven different media: Potato dextrose agar, Carrot agar, Oat meal agar, Water agar, V8 juice agar, Czapek dox agar, and Potato Carrot infusion Agar. With the naked eye, colony features such as colony colour, mycelial growth, and sporulation were investigated. Sporangiophore, monosporous sporangiola, and zygospore were viewed and measured using an Olympus BX53 microscope (Tokyo, Japan) attached with an image analyser.

Study on correlation between weather factors and disease severity

The study investigated meteorological factors influencing the pathogen from disease onset to its disappearance. The relationship between meteorological parameters and the occurrence of Choanephora blight of okra was examined at the experimental plot of IIHR-Central Horticulrural Experiment station, Bhubaneswar. Disease severity data was recorded weekly.

Molecular Characterization

Molecular characterization of the causal fungi isolate CHO-C3 was conducted for further confirmation. Culture of the fungi was grown in potato dextrose broth for 7 days at 28 ± 2°C, and the mycelial mat was carefully extracted while maintaining sterility by passing it through Whatman No. 1 filter paper. After rinsing with sterile distilled water and drying to remove excess moisture, 2 g of dried mycelium was used to extract genomic DNA using the CTAB method. Before the isolated DNA was stored at -20°C for additional analysis, its quality was evaluated on a 0.8% agarose gel. A segment of the ITS rDNA was amplified using specific primers ITS1 and ITS4 (White et al., 1990). PCR amplification was carried out in a 25 µL reaction mixture containing fungal genomic DNA, PCR buffer, MgCl2, dNTPs, primers, Taq DNA polymerase, and nuclease-free water. The amplification process consisted of 35 cycles with denaturation, annealing, and extension steps. Using a gel documentation system, PCR products were electrophoresed in agarose gels, stained with ethidium bromide, and made visible. Amplicons were then purified and sequenced bidirectionally using the ITS1 and ITS4 primers. Resulting sequences were deposited in GenBank, and accession numbers were obtained. Using the Basic Local Alignment Search Tool (BLAST) program available on the National Centre for Biotechnology Information website (www.ncbi.nlm.nih.gov/), the identity of nucleotide sequences was examined.

MEGA version 6.0.1 was used for the phylogenetic analysis (Tamura et al. 2013). For reference, the nucleotide sequence of the fungus's ITS rDNA region was compared to those of reported ITS sequences from the database that belonged to various Choanephora species that infected various hosts. The MEGA software's ClustalW technique was used to align the reference sequences of the nearest type species that belong to the same genus. BioEdit Sequence Alignment Editor, version 5.0.9 (Hall 1999), was used to create the nucleotide sequence identity matrixes for the Choanephora species. With 1,000 bootstrapped replications, the neighbour-joining method was used to build the phylogenetic tree and assess evolutionary distances.

In-vitro bio-efficacy of latest fungicide molecules against the test pathogen

The relative bio-efficacy of latest fungicide molecules were evaluated using the poisoned food technique at various concentrations (Nene and Thapliyal,1993). Eleven fungicides such as, Mancozeb 75% WP (Dhanuka M-45), Copper Oxychloride 50% WP (Blitox50), Carbendazim 50% WP (Kemistin), Captan 50% WP (Captaf), Azoxystrobin 23% WP (Mirador), Tebuconazole 25.9% EC (Tebu), Difenoconazole 25% EC (Score), Copper hydroxide 53.8% DF (Kocide), Hexaconazole 5% EC (Contaf), Thiophenate methyl 70% WP (Roko), Propineb 70% WP (Antracol) and five combination compound fungicides, Tebuconazole 10% + Sulphur 65% WG (Cultivo),Tebuconazole 50% + Trifloxystrobin 25% WG (Nativo), Carbendazim 12% + Mancozeb 63% WP (Saaf), Carboxin 37.5% + Thiram 37.5% WP (Vitavax power), Metiram 55% + Pyraclostrobin 5% WG (Clutch) were tested against C. cucurbitarum. Each fungicide was evaluated at six different concentrations (0.05%, 0.10%, 0.15%, 0.20%, 0.25% and 0.30%) with four replications for each concentration in complete randomized design with suitable control without fungicide. The following formula was used to determine the percent inhibition of mycelial growth (Vincent, 1927).

$$\:Percent\:inhibition\:of\:mycelial\:growth=\frac{Colony\:diameter\:in\:control\left(mm\right)-Colony\:diameter\:in\:treatment\left(mm\right)\:}{Colony\:diameter\:in\:control\:\left(mm\right)}\times\:100$$

Survey and Disease incidence

During the 2019-2020 kharif season, a state wide survey was carried out to six major okra-growing districts of Odisha viz., Jajpur, Cuttack, Khordha, Bhadrak, Balasore, and Mayurbhanj, encompassing thirty villages. The comprehensive findings across all the locations revealed that the disease incidence varying from 0.0 to 35.0 % (Table 1 & Figure 1). Among them, Khordha district exhibited the highest wet rot incidence (25.0 to 35.0%), while Bhadrak reported the lowest (0.0 to 10.0%) (Table 1) wet rot incidence.

Symptomatology

The initial symptoms of the pathogen were first evident in young plants, primarily attacking mature or senescent leaves close to the soil region. Subsequently, the infection spread to remaining leaves turning them to charcoal black with numerous whitish-stalked, black-globular headed fruiting structures emerging from the infected region, resembling black-headed pins (Figure 2a). During the reproductive phase, symptoms first emerged as water-soaked lesions on newly blossomed flowers as blossom blight, resulting in their decay (Figure 2b). Infected flowers hung loosely on the plant and the surviving flowers began developing young pods or fruits, initially showing chlorotic water-soaked lesions, which later progressed to wet rot symptoms with fungal sporulation on the rotted fruits due to pathogen spread from flowers to pods (Figure 2c). Leaf symptoms manifested as brown necrotic regions, spreading from the margin to inwards (Figure 2d). Irrespective of the plant part and stage of infection distinctive symptom of this fungus include white stalked, black-headed sporangiophores protruding like black-headed pins from infected tissues.

Disease severity displayed a positive correlation with atmospheric maximum temperature, rainfall, relative humidity, bright sunshine hours and a negative correlation with minimum temperature (Figure 3). The incidence and severity of the pathogen was favoured by intermittent rainfall (153.3 mm), high relative humidity (96%), bright sunshine hours (8.4 hrs) and elevated temperatures (36 ℃).

Isolation and phenotypic characterization of the pathogen

Plant parts showing symptoms and abundant spore production were gathered to determine the identity of the causative agent. Leaves, leaf stalks, fruits, flower buds, and growing tips exhibiting infection were carefully sampled using a sterile needle and shifted onto a medium of Potato Dextrose Agar (PDA) supplemented with 100 mg/l streptomycin sulphate. These samples were then incubated at a temperature of 25 ± 2℃. Within 24 to 48 hours of incubation, mycelial growth was observed on all of the inoculated plates.

The culture initially appeared white with aerial mycelia, transitioned to pale yellow within 3-4 days (Figure 5a and 5b). The mycelium was hyaline, unbranched, and aseptate (Figure 6a). Occasional sporangiophores containing sporangiola were observed in some 4-5 days old cultures at low temperature (20±2°C), though not a common phenomenon (Figure 6b). Microscopically, sporangiophores resembled stalked dandelion flowers, bearing numerous monosporous sporangiola. These sporangiophores were upright to slightly bent, unbranched, and bore secondary branches with sporangiola at the top (Figure 6c). The indehiscent sporangiola were brown to reddish-brown, ellipsoid to broadly ellipsoid, longitudinally striated, measuring 7.5–12 µm wide and 10–11.3 µm high (Figure 6d). Sporangiospores from sporangia were ellipsoid, brown to reddish-brown, with hyaline appendages at each pole (Figure 6e). On old culture plates Zygospores were seen. Those were dark, thick, blackish, spherical and formed between tips of twining branches (Figure 6f).

Among the seven different media viz Carrot agar (T1), Potato dextrose agar (T2), Oat meal agar (T3), Water agar (T4), V8 juice agar (T5), Czapek dox agar (T6), and Potato Carrot infusion Agar (T7), evaluated Potato Dextrose Agar medium exhibited the highest radial mycelial growth (90 mm), surpassing other media making it as the most suitable media for cultivation of Choanephora cucurbitarum. Conversely, the lowest radial mycelial growth was observed in Czapek dox agar medium (68.38 mm) (Figure 4). The detailed cultural characters of the pathogen in different media are explained in Table 2.

Pathogenicity

A representative test isolate CHO-C3 was selected from the isolates, and pathogenicity tests were conducted on detached leaf assays as well as on whole potted plants, alongside with appropriate controls. For the detached leaf assay, a make shift humid environment with a temperature of 26 ± 2 °C having RH of 80% was created and maintained. Within 24–48 hours of inoculation, lesions with pinhead-like sporangia emerged, while control leaves exhibited no symptoms. The retrieved isolates maintained identical morphological traits to the original isolates, affirming their characteristics. Similarly, pathogenicity assessments were executed on potted okra plants. Sporangiola suspension (2×10⁴ spores/ml) was sprayed until runoff on plants designated as inoculated. Whereas sterile distilled water was sprayed on control plants. After 48 hours of inoculation in a makeshift humid chamber (26 ± 2 °C and 80–90% RH), water-soaked lesions appeared on infected plants, followed by flower and fruit rot 4–5 days later; however, control plants remained no symptoms. The recovered Choanephora isolates exhibited consistent morphological features with the original isolates, confirming Koch's postulates. Additionally, cross-inoculation was conducted on potted chilli plants, mirroring the aforementioned process. Water-soaked lesions with characteristic sporangiola emerged on leaves and fruits of infected plants after 2-3 days of inoculation, while untreated control plants remained unaffected. These experiments were replicated twice with consistent resulting.

Molecular characterization of the pathogen

PCR amplification of the ITS-rDNA region of fungal isolate CHO-C3 resulted in an expected amplicon size of 550 bp, as observed on a 1.2% agarose gel. Nucleotide BLAST analysis of the CHO-C3 isolate (GenBank accession no. ON152865) revealed a 99.78% similarity with C. cucurbitarum (GenBank accession no. MK621200). In a phylogenetic tree constructed using the neighbour-joining method, the C. cucurbitarum isolate from okra formed a distinct clade alongside reference isolates of C. cucurbitarum retrieved from GenBank (Figure 7). Thus, the analysis confirmed that the isolate obtained in this study indeed belonged to the species C. cucurbitarum.

In-vitro bio-efficacy of latest fungicide molecules against C. cucurbitarum

Among the 11 individual fungicide molecules evaluated, tebuconazole, difenoconazole, and hexaconazole exhibited complete (100.0%) inhibition of mycelial growth across all tested concentrations, significantly distinguishing themselves from the other compounds. Conversely, copper oxychloride displayed complete inhibition (100.0%) of mycelial growth from concentration of 0.15% onwards. Captan, azoxystrobin, and propineb demonstrated complete inhibition (100.0%) of test pathogen at concentrations of 0.25% and 0.3%, respectively, remaining consistent with the previously mentioned treatments. Meanwhile, mancozeb, copper hydroxide, and thiophanate methyl fungicides showcased increased inhibition with increasing concentration, attaining peak inhibitions of 93.33%, 90.56%, and 91.67%, respectively, at a concentration of 0.3%. Carbendazim exhibited the least effectiveness at all concentrations against the test pathogen (Figure 8).

Efficacy of various combination compounds (Ready mix) against the test pathogen, two fungicides, namely tebuconazole 10% + sulphur 65% and tebuconazole 50% + trifloxystrobin 25%, recorded complete (100.0%) inhibition of mycelial growth across all concentrations, marking a significant difference from the rest compounds (Figure 9). Nevertheless, the fungicide metiram 55% + pyraclostrobin 5% achieved a maximum inhibition of 100.0% from a concentration of 0.15% onwards, while carboxin 37.5% + thiram 37.5% attained 100.0% mycelial inhibition at a concentration of 0.3%, aligning with the previously mentioned treatments. In contrast, carbendazim 12% + mancozeb 63% showed the lowest inhibition of mycelial growth at all concentrations among the tested combination compounds against C. cucurbitarum.

Among the various pathogens affecting okra, Choanephora blight is a new emerging disease, which may become potential threat due to its wide host range and significant economic impact. This study addresses the increasing concern over destructive potential of Choanephora blight in okra-growing regions of Odisha, Eastern India located in coastal humid belt. Despite its substantial impact on crop yield and quality, limited comprehensive research has been conducted on this pathogen. Even though the disease has been initially observed during July and October 2019 in our experimental plots, its prevalence was observed in major okra-growing regions in the state of Odisha, during systematic survey in major okra growing belts of Odisha. Statewide survey results unveiled the varying level disease incidence, ranging from 0–35%. Among the six districts surveyed Khordha district demonstrated the highest level of disease incidence. The variation in infection rates across locations can be attributed to localized weather pattern and cropping practices. Incidence of Okra Choanephora blossom blight and pod soft rot in okra was recorded to the tune of 40% in Korea (Park et al., 2015), while leaf blight symptoms affected 5–10% in China (Liu et al., 2019). The symptoms incurred substantial reductions in plant height, leaf vitality, and fresh and dry weights (Johnson et al., 2014).

The fungus exhibited a broad spectrum of infectivity, targeting both vegetative and reproductive plant segments. In the early growth stages, it primarily attacked softer plant sections, progressively spreading to other areas. Severe infections culminated in complete plant demise, resulting in a distinct charcoal-black appearance, akin to burnt vegetation. The affliction extended to blossoming flowers, manifesting as blossom blight symptoms and eventual decay. Initially, young pods displayed chlorotic water-soaked lesions, later developing into a state of wet rot. Leaves bore the brunt of the pathogen, turning charcoal black and twisting as they dangled from the plant. Balogun and Babatola (1999) conducted experiments using potted okra plants to examine the impact of C. cucurbitarum at various growth stages. Early inoculations, during planting and seedling emergence, hastened the leaf infection compared to later stages of plant growth. Older seedlings and flowering plants displayed more rotted buds and fruits, leading to increased abortions and fewer fruits.

A typical sign of this fungal intrusion was the emergence of a white-stalked sporangiophore crowned with a mulberry-like black head, akin to a black-headed pin, on the infected tissues surface. Similar symptomatology was documented in other plants such as Amaranthus, eggplant, Dolichos bean, Yard Long bean and Hibiscus syriacus (Das et al., 2018; Kwon et al., 2005; Kurian et al., 2018 and Park et al., 2016). Notably, the disease escalated in its severity from August to October, coinciding with intermittent rainfall, high relative humidity, and elevated temperatures. Dastur (1920) elucidated the pathogen's shift from saprophyte to a facultative parasite, driven by drastic meteorological shifts - notably, heightened relative humidity, wetness, and temperature. This study sheds light on the dynamic interplay between the pathogen and environmental conditions, contributing to a comprehensive understanding of its behaviour and impact.

The expansive host range of Choanephora as documented by Hyde et al. (2014), encompasses a multitude of flowering and vegetable plants. C. cucurbitarum reported to cause twig blight in chilli (Ganesan et al., 2021), leaf blight in hyacinth bean (Das et al., 2017), shoot disease in amaranthus (Ikediugwu, 1981), and fruit rot in butternut squash (Emmanuel et al., 2021). It also induces twig blight in ban tulsi (Das et al., 2018), leaf blight in papaya (Das et al., 2017), seed pod rot in Moringa oleifera (He et al., 2017), and flower and stem blight in Crotalaria spectabilis (Alfenas et al., 2018). Stem rot in quinoa (Sun et al., 2018), Choanephora flower rot on Hibiscus syriacus (Park et al., 2016), soft rot in eggplant (Kwon et al., 2005), blossom blight in teasle gourd (Das et al., 2017), twig blight in green pea (Das et al., 2017), inflorescence blight, and pod rot in dolichos bean (Kurian et al., 2018) are also attributed. Conspicuously, choanephora leads to leaf blight, pod blight, and Choanephora rot in okra (Liu et al., 2019; Hussein and Ziedan, 2012; Park et al., 2015).

Kirk (1984) categorized two Choanephora species as C. cucurbitarum and C. infundibulifera, based on distinctive features of indehiscent sporangiola. He noted that C. cucurbitarum produces ellipsoid sporangiola with conspicuous longitudinal striations, while C. infundibulifera produces non-striated, subglobose to obovoid sporangiola. Our current study aligns with C. cucurbitarum, as observed sporangiola were indehiscent, longitudinally striated, and ellipsoid to broadly ellipsoid, consistent with Kirk's (1984) and Hyde et al.'s (2014) descriptions.

Among the seven media assessed against growth and sporulation, PDA exhibited optimal conditions for the fungus's proliferation, corroborated by the findings of Sangeeta et al. (2018) and Chowdhury et al. (2020) on this test pathogen. Sporangiophore and sporangiola, was noted on 4 to 5-day-old culture plates, albeit not consistently. This occurrence was attributed by researchers to temperature and humidity's pivotal roles in sporangiola production. Barnett and Lilly's (1955) observations align with this, indicating sporulation's dependence on factors such as temperature, relative humidity, carbon dioxide levels, light exposure, darkness, and nutritional provisions, thus lending credence to the present results. Investigation into various culture media with distinct carbon and nitrogen sources highlighted both similarities and disparities in cultural characteristics. The fungus exhibited fluffy mycelial growth on Carrot Agar and Czapek Dox Agar, dense growth on Oat Meal Agar and Water Agar, and rosette appearance on V8 Juice Agar, while PDA showcased aerial mycelia. Colony color transitioned from white to dirty white and eventually yellowish. These findings echo the seminal contributions of Saroj et al. (2012), Singh et al. (2012), and Park et al. (2015), validating the present observations.

Phylogenetic analysis using ITS rDNA sequences using neighbour joining tree method revealed CHO-C3 isolate from okra formed common clade with C. cucurbitarum reference isolates of C. cucurbitarum obtained from GenBank separate from other Choanephoraceae species. Thus, the fungus was identified as Choanephora cucurbitarum. Similarly, Das et. al. (2018) and Pornsuriya (2017) mentioned that the ITS sequences could separate Choanephora cucurbitarum from the other related species.

The management of plant diseases is a critical aspect of ensuring agricultural productivity and food security. Employing fungicides stands as a paramount strategy for curbing fungal pathogens. In this study, we meticulously evaluated the efficacy of widely employed fungicides in India under laboratory condition. Among the tested singular compound fungicides, tebuconazole 25.9% EC, difenoconazole 25% EC, and hexaconazole 5% EC demonstrated exceptional effectiveness against the test pathogen across all examined concentrations. Conversely, carbendazim 50% WP showcased the least efficacy across all concentrations. In the realm of combined compound chemicals, tebuconazole 10% + sulphur 65% WG and tebuconazole 50% + trifloxystrobin 25% WG achieved complete (100.0%) inhibition of mycelia growth across all concentrations. However, carbendazim 12% + mancozeb 63% WP exhibited the least effectiveness against C. cucurbitarum. Notably, triazole-based fungicides, such as propiconazole, triadimenol, and bitertanol, have demonstrated efficacy against pod rot of cowpea and Choanephora blight of winged bean flowers in previous research, aligning with our current findings (George and Girija, 2015; Gunasekera et al., 1989).

Given the widespread occurrence of Choanephora blight across various vegetables and leguminous plants in India developing suitable management strategies is the need of the hour. George and Girija (2015) evaluated nine fungicides against C. cucurbitarum in vitro, including mancozeb, copper hydroxide, copper oxychloride, azoxystrobin, carbendazim, carboxin, propiconazole, captan + hexaconzole, and carbendazim + mancozeb. Their findings highlighted two contact fungicides, mancozeb and copper oxychloride, and two systemic fungicides, propiconazole and carboxin, exhibited complete pathogen inhibition. Additionally, two combination fungicides, captan + hexaconzole and carbendazim + mancozeb, achieved full suppression at recommended and higher doses. However, the promising fungicide molecules or combination products has to be evaluated under field conditions for their efficacy.

In conclusion, this study comprehensively unravelled the identity of causal agent of wet rot of okra as C. cucurbitarum through thorough morphological, molecular and pathogenicity tests. The findings on prevalence of wet rot across the state reflected the impact of climate change, particularly during southwest monsoon under elevated humidity and temperature, on disease severity. The identified effective fungicides demonstrated promising results in laboratory conditions, empowering farmers with actionable solutions, when translated into field conditions. Ongoing research continues to explore resistant cultivars with available eco-friendly management strategies. Our findings hold the potential to enhance our understanding of Choanephora blight, provide insights into its pathogenesis, and offer valuable clues for disease management, ultimately contributing to the sustainability of okra cultivation.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest. The study presented in the manuscript does not involve human or animal subjects. All authors have reviewed the final version of the manuscript and agree to its submission to your journal.

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Emergence of Choanephora Blight in Okra in Eastern India, Disease Severity, Etiology, and Fungicidal Management

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