Rice Phyllosphere Research Articles

Nitrogen fertilization modulates rice phyllosphere functional genes and pathogens through fungal communities

The phyllosphere is a vital yet often neglected habitat hosting diverse microorganisms with various functions. However, studies regarding how the composition and functions of the phyllosphere microbiome respond to agricultural practices, like nitrogen fertilization, are limited. This study investigated the effects of long-term nitrogen fertilization with different levels (CK, N90, N210, N330) on the functional genes and pathogens of the rice phyllosphere microbiome. Results showed that the relative abundance of many microbial functional genes in the rice phyllosphere was significantly affected by nitrogen fertilization, especially those involved in C fixation and denitrification genes. Different nitrogen fertilization levels have greater effects on fungal communities than bacteria communities in the rice phyllosphere, and network analysis and structural equation models further elucidate that fungal communities not only changed bacterial-fungal inter-kingdom interactions in the phyllosphere but also contributed to the variation of biogeochemical cycle potential. Besides, the moderate nitrogen fertilization level (N210) was associated with an enrichment of beneficial microbes in the phyllosphere, while also resulting in the lowest abundance of pathogenic fungi (1.14 %). In contrast, the highest abundance of pathogenic fungi (1.64 %) was observed in the highest nitrogen fertilization level (N330). This enrichment of pathogen due to high nitrogen level was also regulated by the fungal communities, as revealed through SEM analysis. Together, we demonstrated that the phyllosphere fungal communities were more sensitive to the nitrogen fertilization levels and played a crucial role in influencing phyllosphere functional profiles including element cycling potential and pathogen abundance. This study expands our knowledge regarding the role of phyllosphere fungal communities in modulating the element cycling and plant health in sustainable agriculture.

Uncovering microbiomes of the rice phyllosphere using long-read metagenomic sequencing

The plant microbiome is crucial for plant growth, yet many important questions remain, such as the identification of specific bacterial species in plants, their genetic content, and location of these genes on chromosomes or plasmids. To gain insights into the genetic makeup of the rice-phyllosphere, we perform a metagenomic analysis using long-read sequences. Here, 1.8 Gb reads are assembled into 26,067 contigs including 142 circular sequences. Within these contigs, 669 complete 16S rRNA genes are clustered into 166 bacterial species, 121 of which show low identity (<97%) to defined sequences, suggesting novel species. The circular contigs contain novel chromosomes and a megaplasmid, and most of the smaller circular contigs are defined as novel plasmids or bacteriophages. One circular contig represents the complete chromosome of a difficult-to-culture bacterium Candidatus Saccharibacteria. Our findings demonstrate the efficacy of long-read-based metagenomics for profiling microbial communities and discovering novel sequences in plant-microbiome studies.

Effects of Foliar Application of Silicon Fertilizers on Phyllosphere Bacterial Community and Functional Genes of Paddy Irrigated with Reclaimed Water

Agricultural utilization of reclaimed water is considered to be an effective way to solve water shortage and reduce water environmental pollution. Silicon fertilizer can improve crop yield and quality and enhance crop resistance. The effect of foliar spray with silicon fertilizer on phyllosphere microbial communities remains lacking. In this study, a pot experiment was conducted to explore the effects of different types of silicon fertilizer on the composition and diversity of a phyllosphere bacterial community and the abundances of related functional genes in rice irrigated with reclaimed water. The results showed that Firmicutes, Proteobacteria, Actinobacteriota, Bacteroidota, and Verrucomicrobiota dominated the phyllosphere bacteria of rice. The relative abundance of Bacillus was higher than that of other treatments in RIS3. Reclaimed water irrigation significantly increased the relative abundances of the potential pathogens Pantoea and Enterobacter. The unclassified bacteria were also an important part of the bacterial community in the rice phyllosphere. Bacillus, Exiguobacterium, Aeromonas, and Citrobacter were significantly enriched by silicon fertilizer treatments. Functional prediction analysis showed that indicator species were mainly involved in metabolism and degradation functions, and the predicted functional groups of phyllosphere bacteria were attributed to chemoheterotrophy, aerobic chemoheterotrophy, nitrate reduction, and fermentation. Quantitative PCR results showed that AOA, AOB, and nifH genes were at low abundance levels in all treatments, and nirK genes was not significantly different among treatments. These results contribute to the in-depth understanding of the effects of foliar spray silicon fertilizer on the bacterial community structure and diversity of rice phyllosphere and provide a theoretical basis for the application of silicon fertilizer in reclaimed water irrigation agriculture.

Microbiome homeostasis on rice leaves is regulated by a precursor molecule of lignin biosynthesis

In terrestrial ecosystems, plant leaves provide the largest biological habitat for highly diverse microbial communities, known as the phyllosphere microbiota. However, the underlying mechanisms of host-driven assembly of these ubiquitous communities remain largely elusive. Here, we conduct a large-scale and in-depth assessment of the rice phyllosphere microbiome aimed at identifying specific host-microbe links. A genome-wide association study reveals a strong association between the plant genotype and members of four bacterial orders, Pseudomonadales, Burkholderiales, Enterobacterales and Xanthomonadales. Some of the associations are specific to a distinct host genomic locus, pathway or even gene. The compound 4-hydroxycinnamic acid (4-HCA) is identified as the main driver for enrichment of bacteria belonging to Pseudomonadales. 4-HCA can be synthesized by the host plant’s OsPAL02 from the phenylpropanoid biosynthesis pathway. A knockout mutant of OsPAL02 results in reduced Pseudomonadales abundance, dysbiosis of the phyllosphere microbiota and consequently higher susceptibility of rice plants to disease. Our study provides a direct link between a specific plant metabolite and rice phyllosphere homeostasis opening possibilities for new breeding strategies.

Insilico analysis of adenylation and ketosynthase domain related to antifungal activity of rice phyllosphere bacteria

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Antifungal activities of bacteria producing bioactive compounds isolated from rice phyllosphere against Pyricularia oryzae

Antifungal activities of bacteria producing bioactive compounds isolated from rice phyllosphere against Pyricularia oryzae

Distinctive Structure and Assembly of Phyllosphere Microbial Communities between Wild and Cultivated Rice.

Wild rice has been demonstrated to possess enriched genetic diversity and multiple valuable traits involved in disease/pest resistance and abiotic stress tolerance, which provides a potential resource for sustainable agriculture. However, unlike the plant compartments such as rhizosphere, the structure and assembly of phyllosphere microbial communities of wild rice remain largely unexplored. Through amplicon sequencing, this study compared the phyllosphere bacterial and fungal communities of wild rice and its neighboring cultivated rice. The core phyllosphere microbial taxa of both wild and cultivated rice are dominated with Pantoea, Methylobacterium, Nigrospora, and Papiliotrema, which are potentially beneficial to rice growth and health. Compared to the cultivated rice, Methylobacterium, Sphingomonas, Phaeosphaeria, and Khuskia were significantly enriched in the wild rice phyllosphere. The potentially nitrogen-fixing Methylobacterium is the dominated wild-enriched microbe; Sphingomonas is the hub taxon of wild rice networks. In addition, the microbiota of wild rice was more governed by deterministic assembly with a more complicated and stable community network than the cultivated rice. Our study provides a list of the beneficial microbes in the wild rice phyllosphere and reveals the microbial divergence between wild rice and cultivated rice in the original habitats, which highlights the potential selective role of wild rice in recruiting specific microbiomes for enhancing crop performance and promoting sustainable food production. IMPORTANCE Plant microbiota are being considered a lever to increase the sustainability of food production under a changing climate. In particular, the microbiomes associated with ancestors of modern cultivars have the potential to support their domesticated cultivars. However, few efforts have been devoted to studying the biodiversity and functions of microbial communities in the native habitats of ancestors of modern crop species. This study provides a list of the beneficial microbes in the wild rice phyllosphere and explores the microbial interaction patterns and the functional profiles of wild rice. This information could be useful for the future utilization of the plant microbiome to enhance crop performance and sustainability, especially in the framework of sustainable agroecosystems.

Application of data integration for rice bacterial strain selection by combining their osmotic stress response and plant growth-promoting traits.

Agricultural application of plant-beneficial bacteria to improve crop yield and alleviate the stress caused by environmental conditions, pests, and pathogens is gaining popularity. However, before using these bacterial strains in plant experiments, their environmental stress responses and plant health improvement potential should be examined. In this study, we explored the applicability of three unsupervised machine learning-based data integration methods, including principal component analysis (PCA) of concatenated data, multiple co-inertia analysis (MCIA), and multiple kernel learning (MKL), to select osmotic stress-tolerant plant growth-promoting (PGP) bacterial strains isolated from the rice phyllosphere. The studied datasets consisted of direct and indirect PGP activity measurements and osmotic stress responses of eight bacterial strains previously isolated from the phyllosphere of drought-tolerant rice cultivar. The production of phytohormones, such as indole-acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), and cytokinin, were used as direct PGP traits, whereas the production of hydrogen cyanide and siderophore and antagonistic activity against the foliar pathogens Pyricularia oryzae and Helminthosporium oryzae were evaluated as measures of indirect PGP activity. The strains were subjected to a range of osmotic stress levels by adding PEG 6000 (0, 11, 21, and 32.6%) to their growth medium. The results of the osmotic stress response experiments showed that all bacterial strains accumulated endogenous proline and glycine betaine (GB) and exhibited an increase in growth, when osmotic stress levels were increased to a specific degree, while the production of IAA and GA considerably decreased. The three applied data integration methods did not provide a similar grouping of the strains. Especially deviant was the ordination of microbial strains based on the PCA of concatenated data. However, all three data integration methods indicated that the strains Bacillus altitudinis PB46 and B. megaterium PB50 shared high similarity in PGP traits and osmotic stress response. Overall, our results indicate that data integration methods complement the single-table data analysis approach and improve the selection process for PGP microbial strains.

Culturable Screening of Plant Growth-Promoting and Biocontrol Bacteria in the Rhizosphere and Phyllosphere of Wild Rice.

Wild rice is an important improved resource for cultivated rice and its unique ability to resist biotic and abiotic stress has attracted the attention of many scholars. The microbial community structure in the rhizosphere and leaf area of different rice varieties is also different, which may be one of the reasons for the difference in stress resistance between wild rice and cultivated rice. Forty-six bacteria were screened from the rhizosphere and phyllospheric of four different wild rice varieties. The results of functions of the screened strains showed that 18 strains had a good inhibitory effect on rice blast, and 33 strains had the ability to dissolve phosphorus, potassium, or fix nitrogen. Through potted experiment, the three bacterial strains, 499G2 (Peribacillus simplex), 499G3 (Bacillus velezensis), and 499G4 (B. megaterium) have a positive effect on the growth of cultivated rice in addition to the resistance to rice blast. The contents of total nitrogen, total phosphorus, total potassium, indole acetic acid (IAA), and chlorophyll in plant leaves were increased. In addition, in the verification test of rice blast infection, the application of inoculants can significantly reduce the content of malondialdehyde (MDA), increase the content of soluble sugar, and increase the activity of plant antioxidant enzymes, which may thereby improve rice in resisting to rice blast.

Effects of rare earth elements on bacteria in rhizosphere, root, phyllosphere and leaf of soil\u2013rice ecosystem

The effects of rare earth mining on rice biomass, rare earth element (REE) content and bacterial community structure was studied through pot experiment. The research shows that the REE content in rice roots, shoots and grains was significantly positive correlated with that in soil, and the dry weight of rice roots, shoots and grains was highly correlated with soil physical and chemical properties, nutrient elements and REE contents; The exploitation of rare earth minerals inhibited a-diversity of endophytic bacteria in rhizosphere, root, phyllosphere and leaf of rice, significantly reduced the abundance index, OTU number, Chao, Ace index and also significantly reduced the diversity index–Shannon index, and also reduced uniformity index: Pielou’s evenness index, which caused β-diversity of bacteria to be quite different. The exploitation of rare earth minerals reduces the diversity of bacteria, but forms dominant bacteria, such as Burkholderia, Bacillus, Buttiauxella, Acinetobacter, Bradyrhizobium, Candida koribacter, which can degrade the pollutants formed by exploitation of rare earth minerals, alleviate the compound pollution of rare earth and ammonia nitrogen, and also has the function of fixing nitrogen and resisting rare earth stress; The content of soil available phosphorus in no-mining area is lower, and the dominant bacteria of Pantoea formed in such soil, which has the function of improving soil phosphorus availability. Rare earth elements and physical and chemical properties of soil affect the community structure of bacteria in rhizosphere and phyllosphere of rice, promote the parallel movement of some bacteria in rhizosphere, root, phyllosphere and leaf of rice, promote the construction of community structure of bacteria in rhizosphere and phyllosphere of rice, give full play to the growth promoting function of Endophytes, and promote the growth of rice. The results showed that the exploitation of rare earth minerals has formed the dominant endophytic bacteria of rice and ensured the yield of rice in the mining area, however, the mining of mineral resources causes the compound pollution of rare earth and ammonia nitrogen, which makes REE content of rice in mining area significantly higher than that in non-mining area, and the excessive rare earth element may enter the human body through the food chain and affect human health, so the food security in the REE mining area deserves more attention.

Scale-Dependent Effects of Growth Stage and Elevational Gradient on Rice Phyllosphere Bacterial and Fungal Microbial Patterns in the Terrace Field.

The variation of phyllosphere bacterial and fungal communities along elevation gradients may provide a potential link with temperature, which corresponds to an elevation over short geographic distances. At the same time, the plant growth stage is also an important factor affecting phyllosphere microorganisms. Understanding microbiological diversity over changes in elevation and among plant growth stages is important for developing crop growth ecological theories. Thus, we investigated variations in the composition of the rice phyllosphere bacterial and fungal communities at five sites along an elevation gradient from 580 to 980 m above sea level (asl) in the Ziquejie Mountain at the seedling, heading, and mature stages, using high-throughput Illumina sequencing methods. The results revealed that the dominant bacterial phyla were Proteobacteria, Actinobacteria, and Bacteroidetes, and the dominant fungal phyla were Ascomycota and Basidiomycota, which varied significantly at different elevation sites and growth stages. Elevation had a greater effect on the α diversity of phyllosphere bacteria than on that phyllosphere fungi. Meanwhile, the growth stage had a great effect on the α diversity of both phyllosphere bacteria and fungi. Our results also showed that the composition of bacterial and fungal communities varied significantly along elevation within the different growth stages, in terms of both changes in the relative abundance of species, and that the variations in bacterial and fungal composition were well correlated with variations in the average elevation. A total of 18 bacterial and 24 fungal genera were significantly correlated with elevational gradient, displaying large differences at the various growth stages. Soluble protein (SP) shared a strong positive correlation with bacterial and fungal communities (p < 0.05) and had a strong significant negative correlation with Serratia, Passalora, unclassified_Trichosphaeriales, and antioxidant enzymes (R > 0.5, p < 0.05), and significant positive correlation with the fungal genera Xylaria, Gibberella, and Penicillium (R > 0.5, p < 0.05). Therefore, it suggests that elevation and growth stage might alter both the diversity and abundance of phyllosphere bacterial and fungal populations.

Integrated Metabarcoding and Culturomic-Based Microbiome Profiling of Rice Phyllosphere Reveal Diverse and Functional Bacterial Communities for Blast Disease Suppression.

Phyllosphere—the harsh foliar plant part exposed to vagaries of environmental and climatic variables is a unique habitat for microbial communities. In the present work, we profiled the phyllosphere microbiome of the rice plants using 16S rRNA gene amplicon sequencing (hereafter termed metabarcoding) and the conventional microbiological methods (culturomics) to decipher the microbiome assemblage, composition, and their functions such as antibiosis and defense induction against rice blast disease. The blast susceptible rice genotype (PRR78) harbored far more diverse bacterial species (294 species) than the resistant genotype (Pusa1602) that showed 193 species. Our metabarcoding of bacterial communities in phyllomicrobiome revealed the predominance of the phylum, Proteobacteria, and its members Pantoea, Enterobacter, Pseudomonas, and Erwinia on the phyllosphere of both rice genotypes. The microbiological culturomic validation of metabarcoding-taxonomic annotation further confirmed the prevalence of 31 bacterial isolates representing 11 genera and 16 species with the maximum abundance of Pantoea. The phyllomicrobiome-associated bacterial members displayed antifungal activity on rice blast fungus, Magnaporthe oryzae, by volatile and non-volatile metabolites. Upon phyllobacterization of rice cultivar PB1, the bacterial species such as Enterobacter sacchari, Microbacterium testaceum, Pantoea ananatis, Pantoea dispersa, Pantoea vagans, Pseudomonas oryzihabitans, Rhizobium sp., and Sphingomonas sp. elicited a defense response and contributed to the suppression of blast disease. qRT-PCR-based gene expression analysis indicated over expression of defense-associated genes such as OsCEBiP, OsCERK1, and phytohormone-associated genes such as OsPAD4, OsEDS1, OsPR1.1, OsNPR1, OsPDF2.2, and OsFMO in phyllobacterized rice seedlings. The phyllosphere bacterial species showing blast suppressive activity on rice were found non-plant pathogenic in tobacco infiltration assay. Our comparative microbiome interrogation of the rice phyllosphere culminated in the isolation and identification of agriculturally significant bacterial communities for blast disease management in rice farming through phyllomicrobiome engineering in the future.

Tracking and assessment of Puccinia graminis f. sp. tritici colonization on rice phyllosphere by integrated fluorescence imaging and qPCR for nonhost resistance phenotyping

Fluorescence microscopy and qPCR-based pathogen tracking tools were developed to elucidate the growth and development of Puccinia graminis f. sp. tritici on leaves of nonhost plant (rice: Oryza sativa L.). Stem rust race-Puccinia graminis f. sp. tritici 40A was identified using Internal Transcribed Spacer (ITS) sequences and gene sequences coding for hypothetical protein (PGTG_08233_hypothetical protein, HP). A reliable qPCR-based quantitation assay was developed for stem rust fungus exploiting HP primers that yielded 377-bp amplicon in conventional PCR and a forma specialis-specific 146 bp in qPCR with a detection sensitivity of 250-fg genomic DNA. The fungal cell wall N-acetyl-glucosamine was green fluorescence labelled and visualized on propidium iodide-stained nonhost leaf. This technique along with scanning electron microscopy allowed imaging of various developmental structures of Puccinia graminis f. sp. tritici 40A on plant epiphytic and endophytic niches. The microscopy coupled with qPCR-based pathogen load estimation revealed that the nonhost (rice) and host (wheat) phyllosphere surface supported uredospore germination, germ tube formation, hyphal elongation, epiphytic growth, stomatal entry and endophytic growth of fungus in an identical manner. However, the nonhost plant did not show any sign of rusting caused by uredospore production, which instead displayed induced H2O2 accumulation, on leaf. Development of qPCR-based Puccinia quantitation and green fluorescent tag-based qualitative assessment of Puccini proliferation will facilitate nonhost resistance phenotyping not only in rice but also in other nonhost plants of stem rust pathogen.

Suppression of Rice Planthopper Populations by the Entomopathogenic Fungus Metarhizium anisopliae without Affecting the Rice Microbiota.

Entomopathogenic fungi can regulate insect populations and function as crucial biological control agents against insect pests, but their impacts on nontarget microorganisms are poorly understood. In this study, we investigated the potential of the fungal strain Metarhizium anisopliae CQMa421 to control rice planthoppers under field conditions and its effects on rice microbiota. This fungus suppressed rice planthoppers during this period, and its control efficiency was more than 60% 7 days after application and did not significantly differ from that of the chemical treatment except in 2019. Both treatments showed a smaller population of rice planthoppers than the controls. After application, M. anisopliae was maintained on rice plants for approximately 14 days, showing a decreasing trend over time. Furthermore, the results showed that the bacterial and fungal richness (operational taxonomic units) and diversity (Shannon index) did not significantly differ between the fungal treatment and the controls after application. The major bacterial taxa of Proteobacteria (including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria), Actinobacteria, Bacteroidetes, and Cyanobacteria accounted for more than 80% of the bacterial community after fungal application, and the major fungal taxa Ascomycota (including Eurotiomycetes, Dothideomycetes, and Sordariomycetes) and Basidiomycota (including Ustilaginomycetes) represented more than 90% of the fungal community. However, the microbial communities of the rice phyllosphere did not significantly change after entomopathogenic-agent application, indicating that the indigenous microbial communities may adapt to fungal insecticide application. Taken together, the results suggest that this fungal agent has good potential for rice planthopper control with no substantial effects on rice microbial communities.IMPORTANCE Entomopathogenic fungi may be used as crucial biocontrol agents for the control of insect pests, but few effective fungal strains have been reported for the control of the rice planthopper, a major pest of rice. More importantly, the impacts of fungal insecticide application on nontarget microorganisms have not been well evaluated, especially under field conditions. Therefore, in this study, we investigated the effects of the fungal strain M. anisopliae CQMa421 on rice planthopper populations from 2017 to 2019 and evaluated its potential impacts on the microbiota of rice plants after application. The results suggested that this fungal agent has good potential for use in the control of rice planthoppers with no significant effects on rice microbial communities, representing an alternative strategy for the control of rice pests.

Mitigation of drought stress in rice crop with plant growth-promoting abiotic stress-tolerant rice phyllosphere bacteria.

In the search of effective drought-alleviating and growth-promoting phyllosphere bacteria, a total of 44 bacterial isolates were isolated from the leaf surface of drought-tolerant rice varieties, Mattaikar, Nootripattu, Anna R(4), and PMK3, and screened for their abiotic stress tolerance by subjecting their growth medium to temperature, salinity, and osmotic stress. Only eight isolates were found to grow and proliferate under different abiotic stress conditions. These isolates were identified using 16S ribosomal DNA gene sequence and submitted to the NCBI database. All the bacterial isolates were identified as Bacillus sp., except PB24, which was identified as Staphylococcus sp., and these isolates were further screened for plant growth-promoting (PGP) traits such as IAA production, GA production, ACC deaminase activity, and exopolysaccharide production under three different osmotic stress conditions adjusted using polyethylene glycol (PEG 6000). Additionally, mineral solubilization was measured under the normal condition. Bacillus endophyticus PB3, Bacillus altitudinis PB46, and Bacillus megaterium PB50 were found to have multifarious PGP traits. Consecutively, the performance of an individual strain to improve the plant growth was investigated under the osmotic stress (25% PEG 6000) and nonstress condition by inoculating them into rice seeds using hydroponics culture. Furthermore, the drought-alleviating potency of bacterial strains was assessed in the rice plants using pot experiment (-1.2 MPa) through bacterial foliar application during the reproductive stage. Finally, as a result of seed inoculation and foliar spray, the application of B. megaterium PB50 significantly improved the plant growth under osmotic stress, protected plants from physical drought through stomatal closure, and improved carotenoid, total soluble sugars, and total protein content. Metabolites of PB50 were profiled under both stress and nonstress conditions using gas chromatography-mass spectroscopy.

Rice phyllosphere bacteria producing antifungal compounds as biological control agents of blast disease

Abstract. Wiraswati SM, Nawangsih AA, Rusmana I, Wahyudi AT. 2020. Rice phyllosphere bacteria producing antifungal compounds as biological control agents of blast disease. Biodiversitas 21: 1273-1278. In vitro analysis of rice phyllosphere bacteria can be developed as biocontrol agents of blast disease using their antifungal activity. To prove the efficacy of bacterial isolates in reducing the severity of blast disease, a greenhouse experiment was conducted. Furthermore, antifungal compounds produced by potential bacterial isolate were also identified using Thin Layer Chromatography (TLC) and Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry (LC-MS/MS) analysis. The application of seven rice phyllosphere bacterial isolates significantly reduced the severity of blast disease in rice (var: Ciherang). Surprisingly, the isolate STGG 14 is the most effective with 70.83% of blast disease reduction. Isolate STGG 14 has been identified as Bacillus subtilis subsp. subtilis which is known as various bioactive compounds producer. It was confirmed through identification of antifungal compounds produced by isolate STGG 14. The bioautography test shows that crude bioactive compounds from isolate STGG 14 could inhibit P. oryzae race 173. On the further analysis using LC-MS/MS, four bioactive compounds isolated from isolate STGG 14 were identified as moracin C, psoralen, 4,6-dimethyl-3 (4’-hydroxyphenyl) coumarin and xanthotoxin. All identified compounds except moracin C, belong to the coumarin group. According to the previous studies, among all compounds, psoralen and xanthotoxin displayed antifungal activity against several fungal pathogens.

Physiological Adaptation and Plant Growth Promoting Functional Traits of Bacillus altitudinis FD48 under In vitro Osmotic Stress

To develop an osmotolerant microbe, as a bioinoculant to mitigate drought it is vital to understand the impact of osmotic stress on their growth and plant growth promoting functional traits. The present study was aimed to evaluate the physiological adaptations and plant growth-promoting traits of a phyllosphere bacterium Bacillus altitudinis FD48 under osmotic stress conditions. The FD48 strain isolated from rice (cultivar ADT43) phyllosphere obtained from Biocatalysts laboratory, Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore. In vitro bioassay was conducted to evaluate the osmotolerant potentials of FD48. B. altitudinis FD48 grown in LB supplemented with PEG 6000 and grown for 48 hrs. Physiological adaptation to osmotic stress was observed by assessing the osmolytes and free amino acids content produced by FD48 under induced stress. Further the plant growth promoting traits under osmotic stress also asceratined. The growth pattern of FD48 strain decreased with the increase in PEG concentrations. The lower level of osmotic stress enhanced the growth of FD48 but at higher concentration exhibited a decline in growth. Enhanced levels of IAA (25 µg g-1 of protein) and EPS (9.76 mg mg-1 protein) production were recorded in the FD48 strain at lower levels of osmotic stress. Furthermore, an increase in osmotic stress had a deleterious effect on IAA production and ACC deaminase activity while the exopolysaccharide production was enhanced. Growth of FD48 under osmotic stress also increased the accumulation of proline and compatible sugars that will protect the FD48 strain by maintaining the turgor potential of cells and stabilizes the membrane proteins. Hence, the results of our study suggesting that, B. altitudinis FD48 strain has the potential to tolerate osmotic stress and might be used as a newer bio-inoculant for triggering moisture deficit stress resilience in plants.

Diversity of Methylobacterium spp. in the Rice of the Vietnamese Mekong Delta.

The Vietnamese Mekong delta is one of the largest rice-producing areas globally. Methylobacterium spp. are persistent colonizers of the rice plant and exert beneficial effects on plant growth and health. Sixty-one Methylobacterium strains belonging to seven species were predominantly isolated from the phyllosphere of rice cultivated in six Mekong delta provinces. Inoculation tests revealed that some strains exhibited plant growth-promoting activity. Moreover, three strains possessed the novel characteristics of inducing leaf bleaching and killing rice seedlings. These results revealed the complex diversity of Methylobacterium in Mekong delta rice and that healthy and productive rice cultivation requires a proper balance of Methylobacterium.

TRFLP analysis for revealing the diversity of rice phyllosphere bacteria

Wiraswati SM, Wahyudi AT, Rusmana I, Nawangsih AA. 2018. TRFLP analysis for revealing the diversity of rice phyllosphere bacteria. Biodiversitas 19: 1743-1749. Phyllosphere environment of rice plant is usually inhabited by diverse bacteria which mostly contribute beneficial effects to the plant fitness. TRFLP method is a rapid and straightforward method to determine the bacterial diversity of many environments, including rice phyllosphere environment. This study aimed to analyze rice phyllosphere bacterial diversity of healthy rice plant cultivar Ciherang obtained from Sukabumi, Jasinga, and Situgede. The bacterial genomes were amplified and digested with two restriction enzymes, i.e., MspI and BstUI. The bacterial diversity (H’ index) and evenness (E index) were calculated from the peak value. From TRFs analysis, Betaproteobacteria and Pseudomonadales were dominantly found in nearly all samples with different relative abundance. In addition, Alphaproteobacteria and Gammaproteobacteria were also dominant in the several samples. The unique bacteria groups were inhabited in the sample from specific regions with certain growth phase. This finding informs us that the geographical factors might be more influent than the growth phase factor. Furthermore, the bacterial diversity and evenness of the metagenomic approach are higher than cultivation-dependent approach.

Interactive effects of Magnaporthe inoculation and nitrogen doses on the plant enzyme machinery and phyllosphere microbiome of resistant and susceptible rice cultivars.

Severity of plant diseases is often influenced by the availability of nutrients, particularly N; however, its effect on the phyllosphere microbiome in foliar pathogen challenged plants is less investigated in rice. The tripartite interaction among the fungal pathogen (Magnaporthe oryzae), rice cultivars (basmati and non-basmati, blast resistant or susceptible) and nitrogen (N) fertilization (0, 120 and 180N) was investigated. Plant growth, elicitation of defense responses and abundance of microbial members in the rice phyllosphere were monitored using biochemical and molecular methods. In general, photosynthetic pigments were distinct for each cultivar, and optimal N doses led to higher values. The susceptible var. CO-39 and resistant CO-39I exhibited higher contents of photosynthetic pigments and micronutrients such as zinc in leaves in response to N doses. Elicitation of defense and hydrolytic enzymes was significantly influenced by pathogen inoculation and modulated by N doses, but varietal effects were distinct. Scoring indices emphasized the pathogen susceptibility of var. CO-39 and PB-1, which showed almost 40-60% higher values than the resistant cultivars; the interactions of cultivars and N doses was also significant. Characteristic changes were recorded in the abundances of the gene copies, particularly, with an overall increase in the number of cyanobacterial 16S rRNA, and bacterial amoA in pathogen-challenged treatments, while nifH gene copies exhibited a reducing trend with increasing N doses, in the presence or absence of pathogen. The varietal differences in the cause and effect relationships can be valuable in crop protection for more effective foliar application of pesticides or biocontrol agents.