First report of leaf spot of maize caused by Curvularia verruculosa in India.

Curvularia leaf spot affects maize plants worldwide and is commonly caused by Curvularia lunata, C. geniculata, and C. pallescens (Manzar et al. 2022; Manzar et al. 2021; Choudhary et al. 2011). In February 2017, leaf spot symptoms were observed in a Deogaon, (25.74 N, 82.99 E) in Uttar Pradesh, India, with disease incidence of less than 10% of the plants in maize fields. On the leaves and sheaths, variously shaped yellow spots were developed. The spots were 2.5 mm in diameter and frequently grew larger, reaching a diameter of 1 cm. They were encircled by a chlorotic halo with dark borders. The symptomatic tissue showing leaf spots of 10 plants was taken and cut into pieces (4 mm2) then surface sterilized with 1% sodium hypochlorite for 1 min, and rinsed three times with distilled water. The cut leaf tissue was placed on the Petri plate containing potato dextrose agar medium amended with streptomycin sulfate (125 ppm). Then incubated at 25±2°C with a 12-h light and dark period, after 5 days of incubation, five pure cultures were obtained using the hyphal tip technique. The pure culture was incubated at 26±2°C for 10 days. The upper surface of the colony was dark grayish black with fluffy mycelia, and the reverse colony was dark brown. The conidia have three septa, are light brown to dark brown in color, straight to curved, ellipsoidal to fusiform, and have two bigger, darker central cells than terminal cells. On average, conidia are between 27.22 to 31.21 mm long and 10.61 to 12.62 mm wide (n=30). The morphological description is similar to the Curvularia verruculosa morphological traits described by Tandon & Bilgrami (Ellis 1966). Molecular identification was done in addition to supporting morphological identification. The nucleopore GDNA Fungus Kit (Genetix Brand, India) was used to extract the genomic DNA of the E40 isolate. The ITS rDNA region (White et al. 1990) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene (Berbee et al. 1999) were amplified through PCR(Manzar et al., 2022).The amplicons were bidirectional sequenced through the Sanger sequencing method. The similarity percentage of E40 isolate matched 100% with MH859788 (CBS444.70 ) of Curvularia verruculosa strain for ITS, and 100% with LT715824 (CBS150.63) of Curvularia verruculosa strain for gpd after Blastn analysis. The gene sequences were deposited to GenBank and accession no. OR262893 for ITS, and LC773704 for gpd were assigned. As a result, C. verruculosa was determined to be the presumed pathogen by both morphology and molecular characteristics. The pathogenicity of E40 isolate was performed twice by spraying (106 conidia/ml in sterile water) onto the leaves of 25 days old maize plant cv. Kanchan (n = 10). Uninoculated healthy maize plants (n=5) were sprayed only with autoclaved water. All pots are kept in a glass house at 25°C±2°C with 90% relative humidity. After 15 days of pathogen inoculation the foliar spots with chlorotic halo, enlarger upto 1cm, and from these spots the identical fungus was reisolated. The reisolated fungus showed similar morphological characteristics to C. verruculosa. Control plants showed no symptoms. C. verruculosa has been previously reported as a causative agent of leaf spot disease in Common beans (Wei et al., 2022), Cotton (Shirsath et al., 2018). To our knowledge, this is the first report of leaf blight caused by C. verruculosa on maize in India.

Tiny but mighty: metal nanoparticles as effective antimicrobial agents for plant pathogen control.

Metal nanoparticles (MNPs) have gained significant attention in recent years for their potential use as effective antimicrobial agents for controlling plant pathogens. This review article summarizes the recent advances in the role of MNPs in the control of plant pathogens, focusing on their mechanisms of action, applications, and limitations. MNPs can act as a broad-spectrum antimicrobial agent against various plant pathogens, including bacteria, fungi, and viruses. Different types of MNPs, such as silver, copper, zinc, iron, and gold, have been studied for their antimicrobial properties. The unique physicochemical properties of MNPs, such as their small size, large surface area, and high reactivity, allow them to interact with plant pathogens at the molecular level, leading to disruption of the cell membrane, inhibition of cellular respiration, and generation of reactive oxygen species. The use of MNPs in plant pathogen control has several advantages, including their low toxicity, selectivity, and biodegradability. However, their effectiveness can be influenced by several factors, including the type of MNP, concentration, and mode of application. This review highlights the current state of knowledge on the use of MNPs in plant pathogen control and discusses the future prospects and challenges in the field. Overall, the review provides insight into the potential of MNPs as a promising alternative to conventional chemical agents for controlling plant pathogens.

Black rot disease incited by Indian race 1 of Xanthomonas campestris pv. campestris in Brassica juncea L. cv. Pusa Bold in India.

Mustard (Brassica juncea L.) is an important oil seed crop in the Brassicaceae family. It is widely cultivated in India for its edible leaves, oil and medicinal properties. In January 2022, we noticed necrotic symptoms typical black rot disease on Brassica juncea (L.) cv. Pusa Bold grown in Indian Agricultural Research Institute, India. Initially, chlorotic lesions emerged on the leaf margin, which progressed to angular V-shaped necrotic lesions and blackened veins. Disease progression became a necrotic appearance in the leaf results browning and papery leaf texture appeared. The suspected causal agent was isolated from three different diseased plants of Pusa Bold on nutrient sucrose agar medium that formed pale yellow, mucoid, and fluidal colonies. Three representative isolates originated from three different plants were sub-cultured on YGCA medium. These isolates are Gram-negative, oxidase negative, KOH positive, nonfluorescent on King's Medium B agar, and positive for starch hydrolysis test (Schaad and White 1974). The 16S ribosomal RNA gene and avirulence genes - AvrBs1 and AvrGf1 were amplified and sequenced in these three isolates with other Xanthomonas campestris pv. campestris (Xcc) isolates. The DNA sequence analysis revealed that these isolates are within the species of X. campestris. The race 1 specific marker namely xcc-b100_4389 was used to characterized the race by detection of 1090bp fragment respectively from gDNA of Xcc isolates (Rubel et al., 2017). The pathogenicity of these isolates was tested twice on youngest leaves of 30-day-old plants of Pusa Bold to convey Koch postulates. Inoculum of three isolates were prepared in nutrient broth at 28°C for 48-h. The pathogenicity test was conducted by small scissors dipped in a bacterial suspension (~ 108 cfu/ml) to cut leaf near margins at 10 points per leaf and the three youngest leaves per plant with three replications. The number of infected points per leaf and the severity of symptoms were assessed 15 and 30 days after inoculation (Singh et al., 2011; 2016). The chlorotic lesions with V-shaped symptoms were appeared on all inoculated plants after 15 and 30 dpi (days post-inoculation). The bacteria were reisolated from inoculated plants and has the same identity as original isolates by using 16S rRNA, avr genes and race 1 specific marker PCR, thereby confirming Koch's postulates. The bacterial inoculation was repeated and the same symptoms appear. Most of the crucifers are infected with black rot disease e.g., cauliflower, cabbage, Brussels, sprout etc. (Vicente et al., 2001). The nucleotide BLAST analysis of 16S rRNA, AvrBs1, AvrGf1 showed a 100% identity with different Xcc strains reported from Germany (B100; AM920689), Brazil (ATCC 33913; AE008922), India (Xcc-C7; CP077958), France (CFBP 5817; CM002673) and China (8004; CP000050) (Singh et al. 2022). Whilst, the nBLAST analysis of xcc-b100_4389 showed 100% nucleotide identity with Xcc race 1 (B100; AM920689), Germany. The sequences were deposited in GenBank (16S rRNA: OM839780; AvrBs1: OM994397; AvrGf1: OM994398; xcc-b100_4389: OM994399). The XccAK1 strain (ITCCBH_0014) was deposited in Indian Type Culture Collection, ICAR-IARI, New Delhi, India. Presently, it is a first report of necrotic black rot on B. juncea cv. Pusa Bold incited by Xcc race 1, India. Previous research reported the black rot disease on other species of the Brassica genus e.g., B. oleracea, and B. napus in Serbia (Popovic et al., 2013) and Argentina (Gaetan et al., 2005).

Evaluating the efficacy of microbial antagonists in inducing resistance, promoting growth, and providing biological control against powdery mildew in wheat.

This study evaluates the biocontrol efficacy of three bacterial strains (Pseudomonas fluorescens DTPF-3, Bacillus amyloliquefaciens DTBA-11, and Bacillus subtilis DTBS-5) and two fungal strains (Trichoderma harzianum Pusa-5SD and Aspergillus niger An-27) antagonists, along with their combinations at varying doses (5.0, 7.5, and 10.0 g/kg of seeds), against wheat powdery mildew. The most effective dose (10 g/kg seeds) was further analyzed for its impact on induced resistance and plant growth promotion under greenhouse conditions. The study measured defense enzyme activities, biochemical changes, and post-infection plant growth metrics. All tested microbial antagonists at 10 g/kg significantly reduced PM severity, with B. subtilis strain DTBS-5 outperforming others in reducing PM severity and achieving the highest biocontrol efficacy. It was followed by B. amyloliquefaciens strain DTBA-11 and P. fluorescens strain DTPF-3, with the fungal antagonists showing no significant effect. Wheat crops treated with B. subtilis strain DTBS-5 exhibited substantial increases in defense-related enzyme activities and biochemicals, suggesting an induced resistance mechanism. The study found a 45% increase in peroxidase (POD) activity, a 50% increase in catalase (CAT) activity, a 30% increase in phenolic content, and a 25% increase in soluble protein content in the wheat plants treated with microbial antagonists. The study highlights the effectiveness of microbial antagonists, particularly B. subtilis strain DTBS-5, in managing wheat PM through biocontrol, induced resistance, and enhanced plant growth, offering a sustainable alternative to chemical treatments.

Screening microbial inoculants and their interventions for cross-kingdom management of wilt disease of solanaceous crops- a step toward sustainable agriculture.

Microbial inoculants may be called magical bullets because they are small in size but have a huge impact on plant life and humans. The screening of these beneficial microbes will give us an evergreen technology to manage harmful diseases of cross-kingdom crops. The production of these crops is reducing as a result of multiple biotic factors and among them the bacterial wilt disease triggered by Ralstonia solanacearum is the most important in solanaceous crops. The examination of the diversity of bioinoculants has shown that more microbial species have biocontrol activity against soil-borne pathogens. Reduced crop output, lower yields, and greater cost of cultivation are among the major issues caused by diseases in agriculture around the world. It is universally true that soil-borne disease epidemics pose a greater threat to crops. These necessitate the use of eco-friendly microbial bioinoculants. This review article provides an overview of plant growth-promoting microorganisms bioinoculants, their various characteristics, biochemical and molecular screening insights, and modes of action and interaction. The discussion is concluded with a brief overview of potential future possibilities for the sustainable development of agriculture. This review will be useful for students and researchers to obtain existing knowledge of microbial inoculants, their activities, and their mechanisms, which will facilitate the development of environmentally friendly management strategies for cross-kingdom plant diseases.

Biogenic synthesis, characterization, and evaluation of synthesized nanoparticles against the pathogenic fungus Alternaria solani.

In the present study, Trichoderma harzianum culture filtrate (CF) was used as a reducing and capping agent to synthesize silver nanoparticles (Ag NPs) in a quick, simple, cost-effective, and eco-friendly manner. The effects of different ratios (silver nitrate (AgNO3): CF), pH, and incubation time on the synthesis of Ag NPs were also examined. Ultraviolet-visible (UV-Vis) spectra of the synthesized Ag NPs showed a distinct surface plasmon resonance (SPR) peak at 420 nm. Spherical and monodisperse NPs were observed using scanning electron microscopy (SEM). Elemental silver (Ag) was identified in the Ag area peak indicated by energy dispersive x-ray (EDX) spectroscopy. The crystallinity of Ag NPs was confirmed by x-ray diffraction (XRD), and Fourier transform infrared (FTIR) was used to examine the functional groups present in the CF. Dynamic light scattering (DLS) revealed an average size (43.68 nm), which was reported to be stable for 4 months. Atomic force microscopy (AFM) was used to confirm surface morphology. We also investigated the in vitro antifungal efficacy of biosynthesized Ag NPs against Alternaria solani, which demonstrated a significant inhibitory effect on mycelial growth and spore germination. Additionally, microscopic investigation revealed that Ag NP-treated mycelia exhibited defects and collapsed. Apart from this investigation, Ag NPs were also tested in an epiphytic environment against A. solani. Ag NPs were found to be capable of managing early blight disease based on field trial findings. The maximum percentage of early blight disease inhibition by NPs was observed at 40 parts per million (ppm) (60.27%), followed by 20 ppm (58.68%), whereas in the case of the fungicide mancozeb (1,000 ppm), the inhibition was recorded at 61.54%.

Development of Diagnostic Markers and Applied for Genetic Diversity Study and Population Structure of Bipolaris sorokiniana Associated with Leaf Blight Complex of Wheat.

Bipolaris sorokiniana, a key pathogenic fungus in the wheat leaf blight complex, was the subject of research that resulted in the development of fifty-five polymorphic microsatellite markers. These markers were then used to examine genetic diversity and population structure in Indian geographical regions. The simple sequence repeat (SSR) like trinucleotides, dinucleotides, and tetranucleotides accounted for 43.37% (1256), 23.86% (691), and 16.54% (479) of the 2896 microsatellite repeats, respectively. There were 109 alleles produced by these loci overall, averaging 2.36 alleles per microsatellite marker. The average polymorphism information content value was 0.3451, with values ranging from 0.1319 to 0.5932. The loci's Shannon diversity varied from 0.2712 to 1.2415. These 36 isolates were divided into two main groups using population structure analysis and unweighted neighbour joining. The groupings were not based on where the isolates came from geographically. Only 7% of the overall variation was found to be between populations, according to an analysis of molecular variance. The high amount of gene flow estimate (NM = 3.261 per generation) among populations demonstrated low genetic differentiation in the entire populations (FST = 0.071). The findings indicate that genetic diversity is often minimal. In order to examine the genetic diversity and population structure of the B. sorokiniana populations, the recently produced microsatellite markers will be helpful. This study's findings may serve as a foundation for developing improved management plans for the leaf blight complex and spot blotch of wheat diseases in India.

Boosting the Biocontrol Efficacy of Bacillus amyloliquefaciens DSBA-11 through Physical and Chemical Mutagens to Control Bacterial Wilt Disease of Tomato Caused by Ralstonia solanacearum.

Bacterial wilt disease of tomato (Solanum lycopersicum L.), incited by Ralstonia solanacearum (Smith), is a serious agricultural problem in India. In this investigation, chemical mutagenic agents (NTG and HNO2 treatment) and ultraviolet (UV) irradiation have been used to enhance the antagonistic property of Bacillus amyloliquefaciens DSBA-11 against R. solanacearum UTT-25 towards an effective management of tomato wilt disease. The investigation established the fact that maximum inhibition to R. solanacearum UTT-25 was exerted by the derivative strain MHNO2-20 treated with nitrous acid (HNO2) and then by the derivative strain MNTG-21 treated with NTG. The exertion was significantly higher than that of the parent B. amyloliquefaciens DSBA-11. These two potential derivatives viz. MNTG-21, MHNO2-20 along with MUV-19, and a wild derivative strain of B. amyloliquefaciens i.e.,DSBA-11 were selected for GC/MS analysis. Through this analysis 18 major compounds were detected. Among the compounds thus detected, the compound 3-isobutyl hexahydropyrrolo (1,2), pyrazine-1,4-dione (4.67%) was at maximum proportion in the variant MHNO2-20 at higher retention time (RT) of 43.19 s. Bio-efficacy assessment observed a record of minimum intensity (9.28%) in wilt disease and the highest bio-control (88.75%) in derivative strain MHNO2-20-treated plants after 30 days of inoculation. The derivative strain MHNO2-20, developed by treating B. amyloliquefaciens with nitrous acid (HNO2), was therefore found to have a higher bio-efficacy to control bacterial wilt disease of tomato under glasshouse conditions than a wild-type strain.

Chromium Toxicity in Plants: Signaling, Mitigation, and Future Perspectives.

Plants are very often confronted by different heavy metal (HM) stressors that adversely impair their growth and productivity. Among HMs, chromium (Cr) is one of the most prevalent toxic trace metals found in agricultural soils because of anthropogenic activities, lack of efficient treatment, and unregulated disposal. It has a huge detrimental impact on the physiological, biochemical, and molecular traits of crops, in addition to being carcinogenic to humans. In soil, Cr exists in different forms, including Cr (III) "trivalent" and Cr (VI) "hexavalent", but the most pervasive and severely hazardous form to the biota is Cr (VI). Despite extensive research on the effects of Cr stress, the exact molecular mechanisms of Cr sensing, uptake, translocation, phytotoxicity, transcript processing, translation, post-translational protein modifications, as well as plant defensive responses are still largely unknown. Even though plants lack a Cr transporter system, it is efficiently accumulated and transported by other essential ion transporters, hence posing a serious challenge to the development of Cr-tolerant cultivars. In this review, we discuss Cr toxicity in plants, signaling perception, and transduction. Further, we highlight various mitigation processes for Cr toxicity in plants, such as microbial, chemical, and nano-based priming. We also discuss the biotechnological advancements in mitigating Cr toxicity in plants using plant and microbiome engineering approaches. Additionally, we also highlight the role of molecular breeding in mitigating Cr toxicity in sustainable agriculture. Finally, some conclusions are drawn along with potential directions for future research in order to better comprehend Cr signaling pathways and its mitigation in sustainable agriculture.

Multi-Gene Phylogenetic Approach for Identification and Diversity Analysis of Bipolaris maydis and Curvularia lunata Isolates Causing Foliar Blight of Zea mays.

Bipolaris species are known to be important plant pathogens that commonly cause leaf spot, root rot, and seedling blight in a wide range of hosts worldwide. In 2017, complex symptomatic cases of maydis leaf blight (caused by Bipolaris maydis) and maize leaf spot (caused by Curvularia lunata) have become increasingly significant in the main maize-growing regions of India. A total of 186 samples of maydis leaf blight and 129 maize leaf spot samples were collected, in 2017, from 20 sampling sites in the main maize-growing regions of India to explore the diversity and identity of this pathogenic causal agent. A total of 77 Bipolaris maydis isolates and 74 Curvularia lunata isolates were screened based on morphological and molecular characterization and phylogenetic analysis based on ribosomal markers-nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region, 28S nuclear ribosomal large subunit rRNA gene (LSU), D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA), and protein-coding gene-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Due to a dearth of molecular data from ex-type cultures, the use of few gene regions for species resolution, and overlapping morphological features, species recognition in Bipolaris has proven difficult. The present study used the multi-gene phylogenetic approach for proper identification and diversity of geographically distributed B. maydis and C. lunata isolates in Indian settings and provides useful insight into and explanation of its quantitative findings.

Unraveling Microbial Volatile Elicitors Using a Transparent Methodology for Induction of Systemic Resistance and Regulation of Antioxidant Genes at Expression Levels in Chili against Bacterial Wilt Disease.

Microbial volatiles benefit the agricultural ecological system by promoting plant growth and systemic resistance against diseases without harming the environment. To explore the plant growth-promoting efficiency of VOCs produced by Pseudomonas fluorescens PDS1 and Bacillus subtilis KA9 in terms of chili plant growth and its biocontrol efficiency against Ralstonia solanacearum, experiments were conducted both in vitro and in vivo. A closure assembly was designed using a half-inverted plastic bottle to demonstrate plant-microbial interactions via volatile compounds. The most common volatile organic compounds were identified and reported; they promoted plant development and induced systemic resistance (ISR) against wilt pathogen R. solanacearum. The PDS1 and KA9 VOCs significantly increased defensive enzyme activity and overexpressed the antioxidant genes PAL, POD, SOD, WRKYa, PAL1, DEF-1, CAT-2, WRKY40, HSFC1, LOX2, and NPR1 related to plant defense. The overall gene expression was greater in root tissue as compared to leaf tissue in chili plant. Our findings shed light on the relationship among rhizobacteria, pathogen, and host plants, resulting in plant growth promotion, disease suppression, systemic resistance-inducing potential, and antioxidant response with related gene expression in the leaf and root tissue of chili.

Screening and Biocontrol Potential of Rhizobacteria Native to Gangetic Plains and Hilly Regions to Induce Systemic Resistance and Promote Plant Growth in Chilli against Bacterial Wilt Disease.

Plant growth-promoting rhizobacteria (PGPR) is a microbial population found in the rhizosphere of plants that can stimulate plant development and restrict the growth of plant diseases directly or indirectly. In this study, 90 rhizospheric soil samples from five agro climatic zones of chilli (Capsicum annuum L.) were collected and rhizobacteria were isolated, screened and characterized at morphological, biochemical and molecular levels. In total, 38% of rhizobacteria exhibited the antagonistic capacity to suppress Ralstonia solanacearum growth and showed PGPR activities such as indole acetic acid production by 67.64% from total screened rhizobacteria isolates, phosphorus solubilization by 79.41%, ammonia by 67.75%, HCN by 58.82% and siderophore by 55.88%. We performed a principal component analysis depicting correlation and significance among plant growth-promoting activities, growth parameters of chilli and rhizobacterial strains. Plant inoculation studies indicated a significant increase in growth parameters and PDS1 strain showed maximum 71.11% biocontrol efficiency against wilt disease. The best five rhizobacterial isolates demonstrating both plant growth-promotion traits and biocontrol potential were characterized and identified as PDS1-Pseudomonas fluorescens (MN368159), BDS1-Bacillus subtilis (MN395039), UK4-Bacillus cereus (MT491099), UK2-Bacillus amyloliquefaciens (MT491100) and KA9-Bacillus subtilis (MT491101). These rhizobacteria have the potential natural elicitors to be used as biopesticides and biofertilizers to improve crop health while warding off soil-borne pathogens. The chilli cv. Pusa Jwala treated with Bacillus subtilis KA9 and Pseudomonas fluorescens PDS1 showed enhancement in the defensive enzymes PO, PPO, SOD and PAL activities in chilli leaf and root tissues, which collectively contributed to induced resistance in chilli plants against Ralstonia solanacearum. The induction of these defense enzymes was found higher in leave tissues (PO-4.87-fold, PP0-9.30-fold, SOD-9.49-fold and PAL-1.04-fold, respectively) in comparison to roots tissue at 48 h after pathogen inoculation. The findings support the view that plant growth-promoting rhizobacteria boost defense-related enzymes and limit pathogen growth in chilli plants, respectively, hence managing the chilli bacterial wilt.