Emergence of Nigrospora Leaf Blight as Major Biotic Stress in South Eastern Coastal Region of India

 
Biotic and abiotic stresses significantly affect final crop yield (Bobode et al., 2021). The occurrence of Nigrospora Oryzae was found on the debris of various living and dead species and is considered a strong parasite that produces grain spots in rice, sorghum and maize. Very recently a leaf spot disease on Yam was reported by Xinyu et al., (2023) and on Chrysanthemum morifolium reported by Sha et al., (2023), on red elephant grass reported by Han et al., (2020). Depending on the host and environment, it has endophytic and parasitic characteristics. Khuskia oryzae H. Huds is a teleomorph of Nigrospora Oryzae. The monotopic genus Khuskia is found throughout the world, especially in tropical regions and is a member of the family Trichosphaeriaceae of the order Trichosphaeriales. The cosmopolitan nature of diverse species range of Nigrospora makes the pathogen more adaptive with the change in climataic conditions. Many Nigrospora species, including N. osmanthi, N. camelliaesinensis, N. lacticolonia and N. vesicularis, have overlapping morphologies, which causes uncertainty in the taxonomic classification of this genus. 
 
The prolonged survey among different districts of south-eastern Odisha reflects that Nigrospora leaf blight turned to be a major biotic stress for rice cultivation. A severe disease incidence of 45-60% with a critical blast-like appearance makes the disease indistinguishable by visual observation from blast diseases. The present investigation was conducted to find out the emergence of the pathogen and disease severity.

Estimation of disease was calculated by analysing disease time and disease pattern assessment in field condition. Random “Diamond” sampling pattern used to collect the sample data where different parameters like growth stage, date of sowing-transplanting, fertilizer application, soil type, texture, pH and various epidemiological parameters considered. The pathogen was isolated from twenty symptomatic plant samples which was collected from infected rice plant parts of various varieties from 4 different districts of Odisha namely Bhadrak (21.0574°N, 86.4963°E), Cuttak (20.4625°N, 85.8830°E), Gajapati (19.1912°N, 84.1857°E) and Ganjam (19.5860°N, 84.6897°E). Isolation of the pathogen was done on potato dextrose agar media. The cultured plate was maintained at 25±2°C for 24-48 hours.
 
The pathogen as subjected to two different types of pathogenicity tests, as follows-
 
Detached leaf assay, where the fresh and non-infected paddy leaf was artificially wounded and inoculated by placing a single mycelial disc on the wounded surface. Isolated culture was maintained under 25±2°C. The observation was taken from a 24-hour interval basis up to 144 hours (Liu et al., 2021).
 
In-vitro pot inoculation technique was used through spraying of spore suspension. In this investigation 3 quantitative traits like number of unfilled grains, disease incidence (%) and grain yield plant^-1 (g) were considered to check the effectivity in 10 rice genotypes (CO 51, CR1017, MTU1140, MTU7029, Black Rice, Barsha, Gobindabhog, Pratiksha, Niranjana and Rajendra Masuri).
 
Cultural variability study was conducted under different nutritional media like potato dextrose agar, V8 agar, czapekdox agar, pikovaskay agar, different dextrose percentage and under different pH. Growth biology of different dextrose limit (5 gm, 10 gm, 20 gm and 25 gm) and different pH level (4.5, 5.5, 6.5 and 8) were tested. All the observations were taken from 24 hours interval basis upto 144 hours.
 
The CTAB DNA extraction procedure verified the molecular identification of isolate Nigrospora oryzae. It was found that Mucorales’ ITS region served as a suitable barcoding marker. Primer pairs ITS-1 and ITS-4 to amplify the ribosomal DNA’s internal transcribed spacers from the Nigrospora oryzae isolate. Amplification was carried out BIO-RAD MY cycler TM thermal Cycler (BIO-RAD, USA). Almost 528 bp product was extracted by sci Genom pvt limited and submitted through NCBI.
 
Phylogenetic analysis was done by comparative analysis of ITS region by neighbour joining method (among the closely related taxa of different countries derived from NCBI Nigrospora database by MEGA version 6.
 
Seed mortality test was performed by placing different rice germplasm on the periphery of dextrose agar plate and at the center mycelia disc placed. The observation was taken from 24 hours interval basis upto 144 hours by estimating seed mortality and seed infection level criteria.
 
Statistical analysis was performed in SPSS and OPSTAT software.
 

From October to December 2021-2023 leaf blight of Oryza sativa caused by Nigrospora oryzae was observed for the first time at Ranadevi farm of Centurion University of Technology and Management, Paralakhemundi (20.2919°N,  85.8419°E) region of Odisha, India. Survey results revealed with 50-60% disease infestation level. Symptoms primarily appeared on any susceptible part like leaf, panicle etc. It created a brownish eye-shaped structure on the leaf surface and which is comparatively larger than the blast symptom (Fig 1), sometimes mixes up with blast and formed a disease consortium. Symptomatic expression of Nigrospora oryzae was measured on Chrysanthemum morifolium by Sha et al. (2023) and on wild rice by Liu et al., (2021).


 
Twenty fungal isolates were obtained from diseased sections of different rice varieties. Cultural characterization analyzed on the PDA media showed cottony white mycelia at the initial stage which turned to brownish to deep black at maturity (Fig 2). Cultural characterization under different nutritional media is represented in Fig 2.

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The hyphae were smooth and had a color ranging from hyaline to pale brown. They were branched and septate, with a diameter of 2-7.5 μm. Conidia were solitary, with a globose or subglobose shape and had a black, shiny and smooth appearance. They were aseptate and measured 11.5-15.5 x 10-15 μm. The conidia of Nigrospora were deeply pigmented and featured germ slits. Similar findings were observed through the morpho cultural description of (Abass et al., 2014 and Li et al., 2018).
 
Symptomatological expression comes after 7-8 days of post-inoculation. Virulency was estimated by the rapid expression of the symptoms and by observing the necrotic area of the lesion. The experimental trial was repeated thrice and every time same symptomatological appearance and virulency were noticed (Fig 3).

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NO-9 and NO-15 isolates were identified as the most virulent among the tested samples. The fungal culture was preserved using PDA (Fig 2A). Morphological studies were based on morphometric characterization of the present isolates NO-9 and NO-15 (Fig 2C, D) with a comparative study with Nigrospora oryzae. The representative isolates NO-9, NO-15 and herbarium were deposited to the National Fungal Culture Collection (NFCCI) and the Ajrekar Mycological Herbarium (AMH) at the Agharkar Research Institute (ARI) in Pune and gained accession numbers NFCCI NO - 5477, NFCCI NO-5478 and AMH - 10510.
 
The pathogen has the ability to devastate rice crops, causing severe yield losses and threatening food security for millions of people worldwide. Typically, it showed incomplete grain filling, which frequently caused a loss of 5 to 25% of grain weight. Presenting results that uncover comparable findings from concurrent studies of Zhai et al., (2013), Chen et al., (2018), Liu et al., (2021), Obisesan and Ojo (2023).
 
In vitro pot inoculation technique conducted with 10 different rice genotypes resulted with susceptibility and superiority detection among the 15 quantitative traits (Fig 4). Disease incidence (DI) and grain yield per plant (GYP) showed variation, while other characters remain static. Comparative assay under treated and non-treated situations (Table 1 and 2) revealed that Niranjana showed the highest disease incidence (60.03%) and Black Rice showed the lowest disease incidence (29.90%) in the treated plot. On the other hand, Barsha showed the highest GYP value (22.00 g) and Gobindabhog showed the lowest GYP value (14.58 g) in the treated plot. Correspondingly, in the non-treated plot, Gobindabhog showed the lowest disease incidence (4.53%) and Niranjana showed the highest disease incidence (8.47%), with addition to that CR1017 showed the highest GYP value (30.72g) and MTU1140 showed the lowest GYP value (23.53 g).

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The PCR products separated in 1.5% TAE Agarose gel revealed on 550- 600 bp products (Fig 5). Isolated DNA, sequenced through Sanger dideoxy technique and resulted 528 bp and 519 bp sequences were deposited in Gene bank which assigned with the accession number OP592217 and OP592218. Molecular identification (ITS-rDNA) followed by blast search of NO 9 (528 bp) and NO 15 (519 bp) sequences are exactly matches with MH748173 (India) and ON514037 (India) with 99% accuracy with NCBI Nigrospora data base. Phylogenetic analysis was done by plotting the isolates through radial phylogeny and maximum likely hood parsimony test. The result reflects that OP592218 and EU918714 (Chaina) are conspecific on the same branch node. Similarly, OP592217 and MH748173 (India) were in another branch node with the highest similarity and closest. (Fig 6 and 7). The results having the parallel findings of Zhao et al., (2014), Liu et al., (2016), Han et al., (2019), Liu et al., (2021) and Wang et al., (2022).

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Growth biology study of Nigrospora was studied under four different media which presented in Table 3. The highest radial growth of Nigrospora was recorded on PDA (8.73 cm), making it the most favorable medium for the fungus. V8 agar medium showed the next best performance, with a mycelial radial growth of 8.23 cm, followed by Pikovskaya’s agar medium with a growth of 7.4 cm. Czapek’s agar medium displayed the least mycelial radial growth of 4.3 cm (see Fig 8. Detailed result is depicted in Table 4.

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Cultural variability study was conducted under the different dextrose concentrations. Nigrospora demonstrated the highest growth at 20 g dextrose concentration (8.9 cm) followed by 25 g and 10 g dextrose concentration, while the lowest growth was observed at 5 g dextrose concentration (3.9 cm) (Fig 9). Detailed result is depicted in Table 5.

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The seed mortality percentage of Pratiksha and Niranjana variety of rice was found to be nearly 60%. With addition to that Niranjana, Pratiksha and MTU 7029 were the most susceptible variety against Nigrospora found under this research (Fig 10). Detailed result is depicted in Table 6. 

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Growth biology of Nigrospora under different pH range was illustrated through Table 7 where 8.9 cm mycelia growth were found after 120 hours, occurred at pH 6.5. Additionally, pH 7.5 (7.63 cm) also supported favourable growth conditions, while the lowest growth was observed at pH 4.5 (4 cm). However, deviating from pH 6.5, either by lowering or increasing the pH level, led to reduced growth for Nigrospora. Remarkably acidic or alkaline pH levels were found to be unsuitable for the pathogen’s growth and sporulation (Fig 11). These results are consistent with the research of Tyagi and Paudel (2014), both indicating that an optimal pH level for fungus growth and sporulation is 6.0. Moreover, an elevation in pH levels demonstrated a hindering impact on both growth and sporulation.

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The emergence of Nigrospora oryzae is a complex issue that demands a multi-dimensional approach. Previously, the appearance of these mild endophytes was reported in several other crops also but the pathogenic emergence on rice as leaf blight is the first time observed from southeastern coastal region of Odisha. Disease progression is accelerated by an ideal range of temperatures (25-30°C) coupling with high moisture levels (80-90%). Increasing disease severity reversely proportional to the grain yield, whereas other quantitative characters remains static from our present investigation we found Niranjana and MTU 7029 are the most susceptible variety against Nigrospora biotic stress and Black Rice and CR1017 are the most resistant variety.

The authors solemnly affirm that they have no conflicts of interest. They are solely associated with the affiliations stated in their contribution and have no ties to any other academic or research institutions. Furthermore, they declare that they hold no financial or non-financial (personal) interests with any other organization or individual. The content of this research paper is entirely original and any material derived from other sources has been properly cited in the references of this manuscript.