Do mutualistic organisms associated with wild Nicotiana benthamiana plants influence drought tolerance?

In arid regions, such as those in north-western Australia, plants survive under water deficit, high temperatures, intense solar radiation and nutrient-impoverished soils. They employ various morphophysiological and biochemical adaptations including interaction with microbial symbionts. Seed from thirty-two accessions of four Nicotiana species (N. benthamiana, N. occidentalis, N. simulans, and N. umbratica) collected from wild plants from northern Australia, were grown and used to assess their responses to water stress. The original wild host plants were selected because they grow in an extremely hot environment where water supply is often unpredictable, and because they share a close genetic relationship to the international model plant N. benthamiana research accession 4 (RA-4). Under moderate water stress conditions, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, root/shoot ratio, and relative water content of leaves was significantly affected. However, the degree to which the accessions were affected varied considerably. Some accessions of N. simulans, N. benthamiana and N. occidentalis were significantly more affected by water stress than others. There was significant inherent variation between accessions in leaf and shoot tip wilting times. Initial symptom expression (leaf wilting) was significantly delayed in two accessions of N. benthamiana and in one accession of N. umbratica. The least water stress tolerant lines, three accessions each of N. occidentalis and N. simulans exhibited advanced symptoms of water stress (shoot tip wilting) within 14-17 days of cessation of watering. This stage was significantly delayed in three accessions of N. benthamiana and two accessions each of N. occidentalis and N. simulans, which exhibited tip wilting after only 21-24 days. There were variations among the accessions of Nicotiana species on their tolerance to water stress. Plant responses to water stress could not be predicted from their phenotype under well-watered conditions. We evaluated identity, host and tissue association, and geographical distribution of fungal endophytes isolated from above and below-ground tissues of wild plants of three indigenous Australian Nicotiana species. Isolation frequency and α-diversity were significantly higher for root endophyte assemblages than those of stem and leaf tissues. We recorded no differences in endophyte species richness or diversity as a function of sampling location, but did detect differences among different host genotypes and plant tissues. There was a significant pattern of community similarity associated with host genotypes but no consistent pattern of fungal community structuring associated with sampling location and tissue type, regardless of the community similarity measurements used. We developed and evaluated two rapid screening methods to identify fungal endophytes that enhanced water deprivation stress tolerance in seedlings of N. benthamiana RA-4. Sixty-eight endophyte isolates taken from wild Nicotiana plants were co-cultivated with N. benthamiana RA-4 seedlings on either damp filter paper or on an agar medium before being subjected to water deprivation. The longevity of seedlings was compared under association with different fungal isolates and under the two screening methods. The filter paper method was faster and simpler than the agarbased method. Based on results, 17 isolates were selected for further testing under water deprivation conditions while growing in washed river sand in a glasshouse. Only two fungal isolates, one resembling Cladosporium cladosporioides (E-162) and a fungus not closely related to any described species (E-284), significantly enhanced seedling tolerance to moisture deprivation consistently in both in vitro and glasshouse-based tests. Although a strongly significant correlation was observed between any two screening methods, the results of the filter paper test was more strongly reflected (r = 0.757, p< 0.001) in results of the glasshouse-based test, indicating its relative suitability over the agar-based test. In another experiment, the same 17 isolates were inoculated to N. benthamiana plants growing in sand in a glasshouse under nutrient-limiting conditions to test their influence on growth promotion. Isolates resembling C. cladosporioides, Fusarium equiseti, and Thozetella sp. promoted seedling growth, evidenced by increased shoot length and higher biomass than non-inoculated control. The two promising fungal endophytes identified from wild Nicotiana plants, E-162 (C. cladosporioides) and E-284 (an unidentified species) were inoculated to plants of N. benthamiana RA-4 to examine their metabolic response to endophyte colonisation under adequate water and water deficit conditions. We examined leaf metabolites using gas chromatography-mass spectrometry (GCMS) to compare levels of sugars, sugar alcohols, amino acids and other metabolites at various stages of plant growth and stress application. Ninety-three metabolites were detected in leaves, including 20 sugars, 13 sugar alcohols, 21 amino acids, 29 organic and fatty acids and ten other compounds. Endophyte colonization caused significant differential accumulation of 17-21 metabolites when the plants were grown under well-watered conditions. The presence of endophytes under water stress conditions caused differential accumulation of cytosine, diethylene glycol, galactinol, glycerol, heptadecanoate, mannose, oleic acid, proline, rhamnose, succinate, and urea. Accumulation of these metabolites suggests that fungal endophytes influence plants to accumulate certain metabolites under water-stress. Further, the two different endophytes tested caused slightly different accumulation patterns of some metabolites. We evaluated how these two fungal endophytes as well as yellowtail flower mild mottle virus (genus Tobamovirus), influenced water stress tolerance in N. benthamiana RA-4 plants. The water stress tolerance of fungus-inoculated plants correlated with increased plant biomass, relative water content, soluble sugars, soluble proteins, proline content, increased activity of the antioxidant enzymes catalase, peroxidase and polyphenol oxidase, and decreased production of reactive oxygen species and electrical conductivity in plants under water stress. In addition, we found that there was significant differential upregulation of drought-related genes in the fungus-inoculated plants subjected to water stress. Plants inoculated with the virus exhibited a similar response to those plant inoculated with the fungi in terms of increasing plant osmolytes, antioxidant enzyme activity and gene expression. Although the fungus and virus infection similarly increased plant water stress tolerance by influencing plant physiology and gene expression, their presence together in the same plant did not have an additive effect, nor did they decrease water stress tolerance. These findings suggest that both fungi and virus influence plant physiology and gene expression under water stress, and it suggests that there is potential to use endophytic fungi, and perhaps virus, to induce greater tolerance to water stress in agricultural production systems

Host Specificity of Endophytic Fungi from Stem Tissue of Nature Farming Tomato (Solanum lycopersicum Mill.) in Japan

To understand the distribution of the cultivable fungal community in plant tissues from nature farming tomato plants, we sampled plants of seven different tomato cultivars and recovered 1742 fungal isolates from 1895 stem tissues sampled from three sites in Japan. Overall, the isolation frequency was low (3&ndash;13%) and the isolation and colonization frequencies did not vary significantly as a function of the cultivar. The fungi were divided into 29 unique operational taxonomic units (OTUs) with 97% ITS gene sequence identity, the majority of which belong to Ascomycota (99.3%). The dominant genera of cultivable endophytic fungi were Fusarium (45.1%), Alternaria (12.8%), Gibberella (12.0%), and Dipodascus (6.8%). The alpha diversity of the fungal endophytes varied among tomato cultivars. Ordination analysis performed to investigate patterns of endophyte community assemblages on the various cultivars revealed that host cultivars had a significant impact on the endophyte community assemblages in all the study sites. Some of the taxa Fusarium, Alternaria, and Penicillium were found on all cultivars, while few were uniquely present in different cultivars. The dominant taxa may be adapted to the particular microecological and physiological conditions present in tomato stems

Impact of Azospirillum sp. B510 on the Rhizosphere Microbiome of Rice under Field Conditions

There has been increasing attention toward the influence of biofertilizers on the composition of microbial communities associated with crop plants. We investigated the impact of Azospirillum sp. B510, a bacterial strain with nitrogen-fixing ability, on the structure of bacterial and fungal communities within rice plant rhizospheres by amplicon sequencing at two sampling stages (the vegetative and harvest stages of rice). Principal coordinate analysis (PCoA) demonstrated a significant community shift in the bacterial microbiome when the plants were inoculated with B510 at the vegetative stage, which was very similar to the effect of chemical N-fertilizer application. This result suggested that the inoculation with B510 strongly influenced nitrogen uptake by the host plants under low nitrogen conditions. Least discriminant analysis (LDA) showed that the B510 inoculation significantly increased the N2-fixing Clostridium, Aeromonas and Bacillus populations. In contrast, there was no apparent influence of B510 on the fungal community structure. The putative functional properties of bacteria were identified through PICRUSt2, and this hinted that amino acid, sugar and vitamin production might be related to B510 inoculation. Our results indicate that B510 inoculation influenced the bacterial community structure by recruiting other N2-fixing bacteria in the absence of nitrogen fertilizer

Impact of <i>Azospirillum</i> sp. B510 on the Rhizosphere Microbiome of Rice under Field Conditions

There has been increasing attention toward the influence of biofertilizers on the composition of microbial communities associated with crop plants. We investigated the impact of Azospirillum sp. B510, a bacterial strain with nitrogen-fixing ability, on the structure of bacterial and fungal communities within rice plant rhizospheres by amplicon sequencing at two sampling stages (the vegetative and harvest stages of rice). Principal coordinate analysis (PCoA) demonstrated a significant community shift in the bacterial microbiome when the plants were inoculated with B510 at the vegetative stage, which was very similar to the effect of chemical N-fertilizer application. This result suggested that the inoculation with B510 strongly influenced nitrogen uptake by the host plants under low nitrogen conditions. Least discriminant analysis (LDA) showed that the B510 inoculation significantly increased the N2-fixing Clostridium, Aeromonas and Bacillus populations. In contrast, there was no apparent influence of B510 on the fungal community structure. The putative functional properties of bacteria were identified through PICRUSt2, and this hinted that amino acid, sugar and vitamin production might be related to B510 inoculation. Our results indicate that B510 inoculation influenced the bacterial community structure by recruiting other N2-fixing bacteria in the absence of nitrogen fertilizer

Arbuscular Mycorrhizal Fungi Associated with Rice (Oryza sativa L.) in Ghana: Effect of Regional Locations and Soil Factors on Diversity and Community Assembly

Understanding the community composition and diversity of arbuscular mycorrhizal fungi (AMF) in an agricultural ecosystem is important for exploiting their potential in sustainable crop production. In this study, we described the genetic diversity and community structure of indigenous AMF in rain-fed rice cultivars across six different regions in Ghana. The morphological and molecular analyses revealed a total of 15 different AMF genera isolated from rice roots. Rhizophagus and Glomus were observed to be predominant in all regions except the Ashanti region, which was dominated by the genera Scutellospora and Acaulospora. A comparison of AMF diversity among the agroecological zones revealed that Guinea Savannah had the highest diversity. Permutational Multivariate Analysis of Variance (PERMANOVA) analysis indicated that the available phosphorus (AP) in the soil was the principal determining factor for shaping the AMF community structure (p < 0.05). We report, for the first time, AMF diversity and community structure in rice roots and how communities are affected by the chemical properties of soil from different locations in Ghana.Japan Society for the Promotion of SciencePeer Reviewe

Exploring Rice Root Microbiome; The Variation, Specialization and Interaction of Bacteria and Fungi In Six Tropic Savanna Regions in Ghana

We investigated the root microbiomes of rice sampled from six major rice-producing regions in Ghana using Illumina MiSeq high-throughput amplicon sequencing analysis. The result showed that both bacterial and fungal community compositions were significantly varied across the regions. Bacterial communities were shaped predominantly by biotic factors, including root fungal diversity and abundance. In contrast, fungal communities were influenced by abiotic factors such as soil nitrate, total carbon and soil pH. A negative correlation between the diversity and abundance of root fungi with soil nitrate (NO3-) level was observed. It suggested that there were direct and indirect effects of NO3- on the root-associated bacterial and fungal community composition. The gradient of soil nitrate from North to South parts of Ghana may influence the composition of rice root microbiome. Bacterial community composition was shaped by fungal diversity and abundance; whereas fungal community composition was shaped by bacterial abundance. It suggested the mutualistic interaction of bacteria and fungi at the community level in the rice root microbiome. Specific bacterial and fungal taxa were detected abundantly in the &lsquo;Northern&rsquo; regions of Ghana, which were very low or absent from the samples of other regions. The analysis of indicator species suggested that an &lsquo;ecological specialization&rsquo; may have occurred which enabled specific microbial taxa to adapt to the local environment, such as the low-nitrate condition in the Northern regions

DataSheet_1_Exogenous nitric oxide promotes salinity tolerance in plants: A meta-analysis.docx

Nitric oxide (NO) has received much attention since it can boost plant defense mechanisms, and plenty of studies have shown that exogenous NO improves salinity tolerance in plants. However, because of the wide range of experimental settings, it is difficult to assess the administration of optimal dosages, frequency, timing, and method of application and the overall favorable effects of NO on growth and yield improvements. Therefore, we conducted a meta-analysis to reveal the exact physiological and biochemical mechanisms and to understand the influence of plant-related or method-related factors on NO-mediated salt tolerance. Exogenous application of NO significantly influenced biomass accumulation, growth, and yield irrespective of salinity stress. According to this analysis, seed priming and foliar pre-treatment were the most effective methods of NO application to plants. Moreover, one-time and regular intervals of NO treatment were more beneficial for plant growth. The optimum concentration of NO ranges from 0.1 to 0.2 mM, and it alleviates salinity stress up to 150 mM NaCl. Furthermore, the beneficial effect of NO treatment was more pronounced as salinity stress was prolonged (>21 days). This meta-analysis showed that NO supplementation was significantly applicable at germination and seedling stages. Interestingly, exogenous NO treatment boosted plant growth most efficiently in dicots. This meta-analysis showed that exogenous NO alleviates salt-induced oxidative damage and improves plant growth and yield potential by regulating osmotic balance, mineral homeostasis, photosynthetic machinery, the metabolism of reactive oxygen species, and the antioxidant defense mechanism. Our analysis pointed out several research gaps, such as lipid metabolism regulation, reproductive stage performance, C4 plant responses, field-level yield impact, and economic profitability of farmers in response to exogenous NO, which need to be evaluated in the subsequent investigation.</p