A Comprehensive Genome-Wide Investigation of the Cytochrome 71 (OsCYP71) Gene Family: Revealing the Impact of Promoter and Gene Variants (Ser33Leu) of OsCYP71P6 on Yield-Related Traits in Indica Rice (Oryza sativa L.).

The cytochrome P450 (CYP450) gene family plays a critical role in plant growth and developmental processes, nutrition, and detoxification of xenobiotics in plants. In the present research, a comprehensive set of 105 OsCYP71 family genes was pinpointed within the genome of indica rice. These genes were categorized into twelve distinct subfamilies, where members within the same subgroup exhibited comparable gene structures and conserved motifs. In addition, 105 OsCYP71 genes were distributed across 11 chromosomes, and 36 pairs of OsCYP71 involved in gene duplication events. Within the promoter region of OsCYP71, there exists an extensive array of cis-elements that are associated with light responsiveness, hormonal regulation, and stress-related signaling. Further, transcriptome profiling revealed that a majority of the genes exhibited responsiveness to hormones and were activated across diverse tissues and developmental stages in rice. The OsCYP71P6 gene is involved in insect resistance, senescence, and yield-related traits in rice. Hence, understanding the association between OsCYP71P6 genetic variants and yield-related traits in rice varieties could provide novel insights for rice improvement. Through the utilization of linear regression models, a total of eight promoters were identified, and a specific gene variant (Ser33Leu) within OsCYP71P6 was found to be linked to spikelet fertility. Additionally, different alleles of the OsCYP71P6 gene identified through in/dels polymorphism in 131 rice varieties were validated for their allelic effects on yield-related traits. Furthermore, the single-plant yield, spikelet number, panicle length, panicle weight, and unfilled grain per panicle for the OsCYP71P6-1 promoter insertion variant were found to contribute 20.19%, 13.65%, 5.637%, 8.79%, and 36.86% more than the deletion variant, respectively. These findings establish a robust groundwork for delving deeper into the functions of OsCYP71-family genes across a range of biological processes. Moreover, these findings provide evidence that allelic variation in the promoter and amino acid substitution of Ser33Leu in the OsCYP71P6 gene could potentially impact traits related to rice yield. Therefore, the identified promoter variants in the OsCYP71P6 gene could be harnessed to amplify rice yields.

Long-term impact of pulses crop rotation on soil fungal diversity in aerobic and wetland rice cultivation.

Pulse crop rotation in rice cultivation is a widely accepted agronomic practice. Depending upon the water regime, rice cultivation has been classified into wetland and aerobic practices. However, no studies have been conducted so far to understand the impact of pulse crop rotation and rice mono-cropping on fungal diversity, particularly in aerobic soil. A targeted metagenomic study was conducted to compare the effects of crop rotations (rice-rice and rice-pulse) on fungal diversity in wetland and aerobic rice soils. Out of 445 OTUs, 41.80% was unknown and 58.20% were assigned to six phyla, namely Ascomycota (56.57%), Basidiomycota (1.32%), Zygomycota (0.22%), Chytridiomycota (0.04%), Glomeromycota (0.03%), and Blastocladiomycota (0.02%). Functional trait analysis found wetland rice-pulse rotation increased symbiotrophs (36.7%) and saprotrophs (62.1%) population, whereas higher pathotrophs were found in aerobic rice-rice (62.8%) and rice-pulse (61.4%) cropping system. Certain soil nutrients played a major role in shaping the fungal community; Ca had significant (p < 0.05) positive impact on saprotroph, symbiotroph and endophytes, whereas Cu had significant (p < 0.05) negative impact on pathotrophs. This study showed that rice-pulse crop rotation could enhance the saprophytic and symbiotic fungal diversity in wetland and reduce the population of pathogens in aerobic rice cultivation.

Deciphering environmental factors and defense response of rice genotypes against sheath blight disease.

Sheath blight (ShB) is one of the most serious diseases in rice, leading to severe yield losses globally. In our study, we evaluated a total of 63 rice genotypes for resistance against sheath blight disease by artificial inoculation over two seasons under field conditions and studied the weather parameters associated with disease incidence. Based on two years of testing, 23 genotypes were found moderately resistant, 38 were moderately susceptible, and 2 exhibited a susceptible reaction to sheath blight disease. Among the specific four genotypes (IC283139, IC283041, IC283038, and IC283023) of the moderately resistant group exhibited less disease reaction in comparison with check variety Tetep. Further, the correlation of percent disease index (PDI) with weather parameters revealed negative associations between PDI and maximum temperature, minimum temperature, low rainfall and the positive association with maximum relative humidity (RH) suggest that very low temperature or high precipitation might have a negative impact on pathogen establishment. In addition, the sheath blight-linked SSRs were assessed using distance and model-based approaches, results of both the models revealed that genotypes distinguished the resistant population from the susceptible one. From the output of two years of principal component analysis, two genotypes from each group of moderately resistant, moderately susceptible and susceptible were studied for their biochemical reaction against the sheath blight pathogen. The biochemical study revealed that the accumulation of defense and antioxidant enzymes, namely, polyphenol oxidase, peroxidase, total phenol, phenylalanine ammonia-lyase, catalase, and superoxide dismutase, were higher in moderately resistant genotypes, but was observed to be lower in moderately susceptible and susceptible genotypes. The statistical analysis revealed the enzyme activities (defense and antioxidant) exhibited a strong negative correlation with area under the disease progress curve (AUDPC) and influence of weather parameter RH. This demonstrates that the environment factor RH plays a major role in imparting the resistance mechanism by decreasing the enzymes activities and increasing PDI. This study found that the identified novel resistant genotype (IC283139) with purple stem base demonstrated improved resistance against sheath blight infection through a defense response and the use of antioxidant machinery.

Elucidation of the population structure and genetic diversity of Bipolaris oryzae associated with rice brown spot disease using SSR markers.

Brown spot disease, caused by Bipolaris oryzae, is one of the several disastrous diseases affecting rice. The brown spot fungus illustrates substantial pathogenic and genetic variability. To the best of our knowledge, extensive analysis utilizing specific SSR primers for B. oryzae genome is quite inadequate for the population structure and genetic diversity of Indian B. oryzae isolates. A total of 84 brown spot isolates were collected from rice-cultivating areas across southern and eastern Indian states, viz., Tamil Nadu, Andhra Pradesh, Odisha and Chhattisgarh. The pathogenicity and virulence characteristics of these isolates were assessed with the susceptible cultivar CR Dhan 201. Twelve genome-specific SSR markers of B. oryzae warranted the investigation of the population structure and genetic diversity among the isolates. These isolates were categorized based on their disease grade as highly virulent isolates (4 nos.), virulent isolates (8 nos.), moderately virulent isolates (47 nos.) and less virulent isolates (25 nos.). PCR amplification and DNA sequencing confirmed the isolates to be B. oryzae. PCR amplification and DNA sequencing confirmed the isolates to be B. oryzae. The SSR markers produced a total of 35 alleles with 1 to 4 alleles per locus with a gene diversity ranging between 0.00 and 0.687 and a major allele frequency variation of 0.425-0.975. The PIC value ranged from 0.00 to 0.638 having a mean value of 0.34. Cluster analysis technique was applied to group the brown spot isolates into four distinct clusters. Principal coordinate and structure analysis identified two genetic clusters of B. oryzae isolates for individual states with some degree of distinctness complying with their virulence. Analysis of molecular variance revealed more genetic variation within populations and less among populations. The study outcome would expedite the comprehension of genetic diversity of B. oryzae across the southern and eastern states of India. Furthermore, we anticipate its guidance in the development of more effective disease management strategies as well as in the generation of novel resistant varieties through marker-assisted breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03347-4.

Lytic polysaccharide monooxygenases (LPMOs) producing microbes: A novel approach for rapid recycling of agricultural wastes.

Out of the huge quantity of agricultural wastes produced globally, rice straw is one of the most abundant ligno-cellulosic waste. For efficient utilization of these wastes, several cost-effective biological processes are available. The practice of field level in-situ or ex-situ decomposition of rice straw is having less degree of adoption due to its poor decomposition ability within a short time span between rice harvest and sowing of the next crop. Agricultural wastes including rice straw are in general utilized by using lignocellulose degrading microbes for industrial metabolite or compost production. However, bioconversion of crystalline cellulose and lignin present in the waste, into simple molecules is a challenging task. To resolve this issue, researchers have identified a novel new generation microbial enzyme i.e., lytic polysaccharide monooxygenases (LPMOs) and reported that the combination of LPMOs with other glycolytic enzymes are found efficient. This review explains the progress made in LPMOs and their role in lignocellulose bioconversion and the possibility of exploring LPMOs producers for rapid decomposition of agricultural wastes. Also, it provides insights to identify the knowledge gaps in improving the potential of the existing ligno-cellulolytic microbial consortium for efficient utilization of agricultural wastes at industrial and field levels.

Understanding rice growth-promoting potential of Enterobacter spp. isolated from long-term organic farming soil in India through a supervised learning approach.

This study addresses the plant beneficial enterobacteria present in rice rhizosphere and their efficiency for enhancing nitrogen uptake in rice plant. Using culturable approaches, the population of total diazotrophs present in rhizosphere samples collected from different organic rice fields of Sikkim were studied and recorded in the range between 4.62 to 4.97 log 10 CFU/g soil. All the isolated commonly occurred diazotrophic bacterial isolates were screened based on their ability to fix nitrogen in milligram per gram of sugar consumed under in-vitro condition with the reference check. In addition to nitrogen fixation, plant growth promoting traits such as production of indole-3-acetic acid and gibberellic acid were estimated using spectrophotometric approaches and compared against Bacillus subtilis as reference multi-potent plant growth promoting strain. In-vivo evaluation of these diazotrophic species in rice found improvement in both above and below ground responses in rice plant evaluated by estimating changes in chlorophyll concentration, plant biomass, root architecture, nitrogen uptake, microbial biomass and associated biochemical activity of soil. Further, the selected isolates were identified through DNA targeted analysis of 16S rRNA gene present in diazotrophs and which identified that the isolates belonged to the Enterobacter genus. Statistical models were prepared for deciphering the dynamics of plant growth improvement due to selective enrichment of rhizosphere bacteria and found significant (p<0.05) correlation between soil and plant parameters. This study concludes that Enterobacter spp. present in organic paddy soils of Sikkim having good nitrogen fixing abilities and whose selective enrichment in rhizosphere improved nitrogen uptake and plant growth promotion in rice plant.

First report of Fusarium proliferatum causing Sheath Rot Disease of Rice in Eastern India.

Sheath rot is one of the most devastating diseases of rice because of its ability to reduce the yield significantly in all rice cultivating areas of the world (Bigirimana et al., 2015). Sheath rot disease is associated with various pathogens such as Sarocladium oryza, Fusarium fujikuroi complex and Pseudomonas fuscovaginae (Bigirimana et al., 2015). Hence, this disease has become more complex in nature and added more seriousness. From September to December 2018, plants were observed with typical sheath rot symptoms in research farm of ICAR-National Rice Research Institute and ten farmer's fields of Cuttack district, Odisha, Eastern India. About 25 to 37% of sheath rot disease severity was recorded in the infected field. Diseased plants were observed with symptoms such as brownish or reddish brown irregular lesions, which were later, got enlarged with grayish centers. Further, rotting of the topmost leaf sheaths that surround the young panicle was observed. At the severe stages, the young panicle was partially emerged from sheath or completely rotted within the sheath. The white to pinkish powdery growth observed inside the infected sheath leading to chaffy and discolored grains. The sheath rot symptomatic plants were collected from the infected fields. To isolate the causal pathogen, infected sheath tissues were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed three times in sterile distilled water, and placed on potato dextrose agar medium (PDA) (HiMedia). Plates were incubated at 27 ± 1° C for 3 d. Further, fungal pathogen colonies were sub-cultured and purified to perform the pathogenicity test. On PDA, the colonies produced abundant white aerial mycelium with violet to pink pigmentation and hyphae were hyaline with septation. Abundant single celled, oval shaped microcondia (5.5-9 × 1.5-2 µm) were produced, whereas macrocondia were not produced and the fungal pathogen was tentatively identified as Fusarium sp. In order to characterize the pathogen at molecular level, ITS, alpha elongation factor gene (EF1-α), RNA polymerase II largest-subunit gene (RPB2), calmodulin gene (cld) were amplified using the primer pair of ITS1/ITS4, EF1/EF2, 5F/7CR and CLPRO1/CLPRO2 respectively and PCR amplicons were subjected to sequencing (White et al. 1990; O'Donnell et al. 1998; Chang et al. 2015). Furthermore, a species-specific primer Fp3-F/Fp4-R was used to identify the pathogen (Jurado et al., 2006). The resulting sequences were confirmed by BLAST analysis and the FUSARIUM-ID database (http://isolate.fusariumdb.org). BLASTn search showed 100% similarity between the query sequence and ITS, EF1-α, RPB2, Calmodulin gene sequences of F. proliferatum available in the Genbank. The following GenBank accession numbers were obtained; MT394055 for ITS; MT439867 for EF1-α; MT790774 for calmodulin; MT940224 for RPB2 and MT801050 for species-specific to F. proliferatum. To confirm the pathogenicity under glass house conditions, fungus grown on sterilized chaffy grains were placed in between boot leaf sheath and panicle and covered with moist cotton (Saravanakumar et al., 2009). After 15 days post inoculation (dpi), rotting symptoms were observed and these were similar to that of field symptoms. Pathogen was constantly re-isolated from symptomatic tissue, satisfying Koch's postulates. Disease symptoms were not observed on un-inoculated plants. Morphological characters, pathogenicity test and molecular characterization have identified the pathogen as F. proliferatum. To the best of our knowledge, this is the first confirmed report of F. proliferatum causing sheath rot disease on rice from Eastern India.

Antagonistic and plant-growth promoting novel Bacillus species from long-term organic farming soils from Sikkim, India.

Three bacteria namely Bacillus luciferensis K2, Bacillus amyloliquefaciens K12 and Bacillus subtilis BioCWB possessing plant growth promotion and biocontrol potential against phytopathogens and rice leaf folder were identified from organic soils of Sikkim, India. The results revealed significant higher production of phytohormones IAA (97.1 µg mL-1) and GA3 (10.6 µg mL-1) was found in K2, whereas BioCWB had higher phosphate solubilization (570.0 µg mL-1) efficacy and also possessed nitrogen fixation ability (5.34 log copy number mL-1 culture). All these bacteria had higher antagonistic activities against phytopathogens viz. Rhizoctonia solani, Fusarium proliferatum, Athelia rolfsii and Colletotrichum gloeosporioides and also had higher larvicidal activity against rice leaf folder Cnaphalocrocis medinalis (Guenne) under in vitro conditions. Molecular insights into the antagonistic mechanisms of Bacillus strains deciphered the presence of several antimicrobial peptides (ericin, subtilin, surfactin, iturin, bacilysin, subtilosin, fengycin and bacillomycin), volatiles (dimethyl disulphide, methyl-Furan, acetic acid, Z-1,3-pentadiene and 3-hexyn-2-ol) and soluble metabolites (9-octadecenamide, E-15-heptadecenal, E-3-eicosene and 5-octadecene). Furthermore, liquid microbial inoculum prepared using the bacterial strains (K2, K12 and BioCWB) were evaluated under glass house (rice) and field condition (capsicum), which significantly enhanced plant growth in rice and yield in capsicum compared to control. The present study revealed the combination of Bacillus spp. (K2, K12 and BioCWB) can be used as bio-inoculants for improving agricultural production in Sikkim. Moreover, for the first time, we demonstrated plant growth promoting (PGP) traits, antifungal and insecticidal properties of B. luciferensis.

Candidate screening of blast resistance donors for rice breeding.

Rice blast is one of the most serious diseases in the world. The use of resistant cultivars is the most preferred means to control this disease. Resistance often breaks down due to emergence of new races; hence identification of novel resistance donors is indispensable. In this study, a panel of 80 released varieties from National Rice Research Institute, Cuttack was genotyped with 36 molecular markers that were linked to 36 different blast resistance genes, to investigate the varietal genetic diversity and molecular marker-trait association with blast resistance. The polymorphism information content of 36 loci varied from 0.11 to 0.37 with an average of 0.34. The cluster analysis and population structure categorized the 80 National Rice Research Institute released varieties (NRVs) into three major genetic groups. The principal co-ordinate analysis displays the distribution of resistant and moderately resistant NRVs into different groups. Analysis of molecular variance result demonstrated maximum (97%) diversity within populations and minimum (3%) diversity between populations. Among tested markers, two markers (RM7364 and pi21_79-3) corresponding to the blast resistance genes (Pi56(t) and pi21) were significantly associated and explained a phenotypic variance of 4.9 to 5.1% with the blast resistance. These associated genes could be introgressed through marker-assisted to develop durable blast resistant rice varieties. The selected resistant NRVs could be good donors for the blast resistance in rice crop improvement research.

Correction: Blast resistance in Indian rice landraces: Genetic dissection by gene specific markers.

[This corrects the article DOI: 10.1371/journal.pone.0211061.].

Blast resistance in Indian rice landraces: Genetic dissection by gene specific markers.

Understanding of genetic diversity is important to explore existing gene in any crop breeding program. Most of the diversity preserved in the landraces which are well-known reservoirs of important traits for biotic and abiotic stresses. In the present study, the genetic diversity at twenty-four most significant blast resistance gene loci using twenty-eight gene specific markers were investigated in landraces originated from nine diverse rice ecologies of India. Based on phenotypic evaluation, landraces were classified into three distinct groups: highly resistant (21), moderately resistant (70) and susceptible (70). The landraces harbour a range of five to nineteen genes representing blast resistance allele with the frequency varied from 4.96% to 100%. The cluster analysis grouped entire 161 landraces into two major groups. Population structure along with other parameters was also analyzed to understand the evolution of blast resistance gene in rice. The population structure analysis and principal coordinate analysis classified the landraces into two sub-populations. Analysis of molecular variance showed maximum (93%) diversity within the population and least (7%) between populations. Five markers viz; K3957, Pikh, Pi2-i, RM212and RM302 were strongly associated with blast disease with the phenotypic variance of 1.4% to 7.6%. These resistant landraces will serve as a valuable genetic resource for future genomic studies, host-pathogen interaction, identification of novel R genes and rice improvement strategies.

Antibiotic-producing Pseudomonas fluorescens mediates rhizome rot disease resistance and promotes plant growth in turmeric plants.

Rhizome rot of turmeric caused by Pythium aphanidermatum is a major threat to turmeric-cultivating areas of India. This study intends to evaluate the performance of fluorescent pseudomonads against Rhizome rot disease and understand the resistance mechanism in Turmeric plants. Fluorescent pseudomonads were screened against Pythium aphanidermatum using dual culture. Selected strains were evaluated for the performance of growth promoting attributes and the presence of antibiotic genes through PCR analysis. Strain FP7 recorded the maximum percent inhibition of P. aphanidermatum under in vitro conditions. Strains FP7 and TPF54 both increased plant growth in turmeric plants in vitro. Strain FP7 alone contained all the evaluated antibiotic biosynthetic genes. Talc and liquid-based formulations were prepared with effective strain and tested for its biocontrol activities under both glasshouse and field conditions. Enzymatic activities of the induced defense enzymes such as PO, PPO, PAL, CAT and SOD were estimated and subjected to spectrophotometric analysis. A combination of rhizome dip and soil drench of FP7 liquid formulation treatment remarkably recorded the minimum disease incidence, higher defense enzymes, maximum plant growth and yield under glasshouse and field conditions. Application of strain FP7 increased the defense molecules, plant growth and yield in turmeric plants thereby reducing the incidence of rhizome rot disease. Moreover, this study has a potential to be adopted for sustainable and eco-friendly turmeric production.