Determination of abundance and symbiotic effectiveness of native rhizobia nodulating soybean and other legumes in Rwanda.

Rhizobia diversity in the rhizosphere is one of the key promoters of biological nitrogen fixation between host legumes and microsymbionts, although related complex interaction may depend on various factors. This research was intended to assess the abundance of indigenous rhizobia isolates under various soil conditions, as well as their effectiveness to nodulate legumes such as soybeans. Factors such as soil properties and legume species influence the volume and symbiotic effectiveness of native rhizobia to nodulate crop legumes. To investigate the abundance of rhizobia isolates, legume crops were uprooted to obtain nodules for most probable number (MPN) determination of rhizobia isolates, and soybean (Glycine max.) was used to verify the presence of suitable and efficient rhizobia strains for nitrogen fixation. Soil samples were obtained from the holes out of which nodules were collected, and the laboratory analysis included pH, Mg, K, available P, organic C, Ca, and N to establish the correlation between the soil status and number of rhizobia isolates' cells. Significant variations (p-value <.05) were observed in the cell counts of Rhizobia isolates from Glycine max, Phaseolus vulgaris, Pisum sativum, and Vigna unguiculata, particularly when compared to Arachis hypogaea isolates under acidic conditions. Notably, Pisum sativum and Vigna unguiculata showed consistent performance across all pH conditions. The number of rhizobia isolates was found to be significantly linked to total N and P deficiencies (p < .05). It was also established that total N was dependent on the number of rhizobia cells and that there is a strong correlation between organic carbon and N content. This study highlights the crucial role of understanding and optimizing conditions for rhizobia nodulation in diverse soil environments, emphasizing its potential impact on enhancing biological nitrogen fixation in legumes.

Integrated Strategies for Durable Rice Blast Resistance in Sub-Saharan Africa.

Rice is a key food security crop in Africa. The importance of rice has led to increasing country-specific, regional, and multinational efforts to develop germplasm and policy initiatives to boost production for a more food-secure continent. Currently, this critically important cereal crop is predominantly cultivated by small-scale farmers under suboptimal conditions in most parts of sub-Saharan Africa (SSA). Rice blast disease, caused by the fungus Magnaporthe oryzae, represents one of the major biotic constraints to rice production under small-scale farming systems of Africa, and developing durable disease resistance is therefore of critical importance. In this review, we provide an overview of the major advances by a multinational collaborative research effort to enhance sustainable rice production across SSA and how it is affected by advances in regional policy. As part of the multinational effort, we highlight the importance of joint international partnerships in tackling multiple crop production constraints through integrated research and outreach programs. More specifically, we highlight recent progress in establishing international networks for rice blast disease surveillance, farmer engagement, monitoring pathogen virulence spectra, and the establishment of regionally based blast resistance breeding programs. To develop blast-resistant, high yielding rice varieties for Africa, we have established a breeding pipeline that utilizes real-time data of pathogen diversity and virulence spectra, to identify major and minor blast resistance genes for introgression into locally adapted rice cultivars. In addition, the project has developed a package to support sustainable rice production through regular stakeholder engagement, training of agricultural extension officers, and establishment of plant clinics.

Pathobiomes Revealed that Pseudomonas fuscovaginae and Sarocladium oryzae Are Independently Associated with Rice Sheath Rot.

Rice sheath rot has been mainly associated with the bacterial pathogen Pseudomonas fuscovaginae and in some cases to the fungal pathogen Sarocladium oryzae; it is yet unclear if they are part of a complex disease. The bacterial and fungal community associated with rice sheath rot symptomatic and asymptomatic rice plants was determined/studied with the main aim to shed light on the pathogen(s) causing rice sheath rot. Plant samples were collected from different rice varieties in two locations (highland and lowland) in two rice-growing seasons (wet and dry season) in Burundi. Our results showed that the bacterial Pseudomonas genus was prevalent in highland in both rice-growing seasons and was not affected by rice plant varieties. Pseudomonas sequence reads displayed a significant high similarity to Pseudomonas fuscovaginae indicating that it is the causal agent of rice sheath rot as previously reported. The fungal Sarocladium genus was on the other hand prevalent in lowland only in the wet season; the sequence reads were most significantly similar to Sarocladium oryzae. These studies showed that plant microbiome analysis is very useful in determining the microorganisms involved in a plant disease. P. fuscovaginae and S. oryzae were prevalent in symptomatic samples in highland and lowland respectively being present independently and hence are not part of a complex disease. The significant presence of other bacterial and fungal taxa in symptomatic samples is also discussed possibly making this disease more complex. Finally, we also report the microbial communities that are associated with the plant sheath in symptomatic and asymptomatic plants from the same rice fields.

The broad-spectrum rice blast resistance (R) gene Pita2 encodes a novel R protein unique from Pita.

BACKGROUND: Rice blast is generally considered the most devastating rice disease worldwide. The development of resistant varieties has been proven to be the most economical strategy to control the disease. A cluster of resistant (R) genes on rice chromosome 12 including Pita, Pita2 and Ptr has been studies for decades. However, the relationship between these R genes has not been well established. RESULTS: In this study, we compared the resistance spectra controlled by Pita2 and Pita by testing their monogenic lines (MLs) in four hotspots found in the Philippines and Burundi from 2014 to 2018. The reaction patterns were distinct in two countries and that Pita2-mediated field resistance was relatively prevalent. Pathogenicity tests using 328 single-spore isolates in greenhouse further verified that IRBLta2-Re for Pita2 conferred a relatively broader spectrum resistance than those of Pita. Rough and fine mapping of Pita2 were conducted using F2 and F3 populations derived from IRBLta2-Re [CO] and CO 39 consisting of 4344 progeny to delimit Pita2 in a genomic interval flanked by two markers 12 g18530 and 12 g18920 proximal to the centromere of chromosome 12. Alignment of the markers to the genomic sequence of IR64, which harbors Pita2 verified by genetic analysis, approximately delimited the candidate gene(s) within 313-kb genomic fragment. The two Pita2 suppressive mutants that contain mutations within Pita2 were verified and identified. Comparative sequence analysis in these two mutants further identified that each individual allele contains a single nucleotide substitution at a different position resulting in nonsense and missense mutations in the protein product of LOC_Os12g18729. On the contrary, no sequence mutation was detected in other candidate genes, indicating that mutations in LOC_Os12g18729 were responsible for the loss of function of Pita2. Pita2 encodes a novel R protein unique from Pita, which is exactly identical to the previously cloned Ptr. Moreover, based on the resistance gene analysis of rice varieties and mutants containing Pita, it was found that Pita2 rather than Pita was responsible for the specificity to some differential isolates with AvrPita. The diagnosis and survey of Pita2 in IRRI released varieties showed relatively low frequency, implying a high value of its application for breeding resistant varieties against rice blast via marker assisted selection. CONCLUSION: Our study clarified the relationship between Pita, Pita2 and Ptr. Pita2 is identical to Ptr and distinct from Pita in both sequence and chromosomal location although Pita2 and Pita are genetically linked to each other. The loss of function of Pita2 but not Pita eliminate the specificity to some AvrPita containing isolates, however, the mechanism underlying the recognition between Pita2/Pita and AvrPita remains elusive.

The plant pathogen Pseudomonas fuscovaginae contains two conserved quorum sensing systems involved in virulence and negatively regulated by RsaL and the novel regulator RsaM.

Pseudomonas fuscovaginae is a Gram-negative fluorescent pseudomonad pathogenic towards several plant species. Despite its importance as a plant pathogen, no molecular studies of virulence have thus far been reported. In this study we show that P. fuscovaginae possesses two conserved N-acyl homoserine lactone (AHL) quorum sensing (QS) systems which we designated PfsI/R and PfvI/R. The PfsI/R system is homologous to the BviI/R system of Burkholderia vietnamiensis and produces and responds to C10-HSL and C12-HSL whereas PfvI/R is homologous to the LasI/R system of Pseudomonas aeruginosa and produces several long-chain 3-oxo-HSLs and responds to 3-oxo-C10-HSL and 3-oxo-C12-HSL and at high AHL concentrations can also respond to structurally different long-chain AHLs. Both systems were found to be negatively regulated by a repressor protein which was encoded by a gene located intergenically between the AHL synthase and LuxR-family response regulator. The pfsI/R system was regulated by a novel repressor designated RsaM while the pfvI/R system was regulated by both the RsaL repressor and by RsaM. The two systems are not transcriptionally hierarchically organized but share a common AHL response and both are required for plant virulence. Pseudomonas fuscovaginae has therefore a unique complex regulatory network composed of at least two different repressors which directly regulate the AHL QS systems and pathogenicity.

Xanthomonas oryzae pv. oryzae XKK.12 contains an AroQgamma chorismate mutase that is involved in rice virulence.

Chorismate mutase (CM) is a key enzyme in the shikimate pathway which is responsible for the synthesis of aromatic amino acids. There are two classes of CMs, AroQ and AroH, and several pathogenic bacteria have been reported to possess a subgroup of CMs designated AroQ(gamma). These CMs are usually exported to the periplasm or outside the cell; in a few cases, they have been reported to be involved in virulence and their precise role is currently unknown. Here, we report that the important rice pathogen Xanthomonas oryzae pv. oryzae XKK.12 produces an AroQ(gamma) CM which we have purified and characterized from spent supernatants. This enzyme is synthesized in planta and X. oryzae pv. oryzae knock-out mutants are hypervirulent to rice. The role of this enzyme in X. oryzae pv. oryzae rice virulence is discussed.

The presence, type and role of N-acyl homoserine lactone quorum sensing in fluorescent Pseudomonas originally isolated from rice rhizospheres are unpredictable.

In Gram-negative bacteria, a typical quorum-sensing (QS) system involves the production and response to N-acyl homoserine lactones (AHLs). It still remains unclear as to how pivotal and conserved AHL QS is in root-colonizing rhizosphere Pseudomonas. We, therefore, performed a systematic study of AHL QS on a set of 50 rice rhizosphere Pseudomonas isolates. We also isolated the AHL QS genes in two representative strains and analyzed the role of AHL QS regulation of various phenotypes. Our results are discussed with the current knowledge of AHL QS of rhizosphere Pseudomonas, implicating a lack of conservation and an unpredictable role played by AHL QS in this group of bacteria.

Involvement of a quorum-sensing-regulated lipase secreted by a clinical isolate of Burkholderia glumae in severe disease symptoms in rice.

Burkholderia glumae is an emerging rice pathogen in several areas around the world. Closely related Burkholderia species are important opportunistic human pathogens for specific groups of patients, such as patients with cystic fibrosis and patients with chronic granulomatous disease. Here we report that the first clinical isolate of B. glumae, strain AU6208, has retained its capability to be very pathogenic to rice. As previously reported for rice isolate B. glumae BGR1 (and also for the clinical isolate AU6208), TofI or TofR acyl homoserine lactone (AHL) quorum sensing played a pivotal role in rice virulence. We report that AHL quorum sensing in B. glumae AU6208 regulates secreted LipA lipase and toxoflavin, the phytotoxin produced by B. glumae. B. glumae AU6208 lipA mutants were no longer pathogenic to rice, indicating that the lipase is an important virulence factor. It was also established that type strain B. glumae ATCC 33617 did not produce toxoflavin and lipase and was nonpathogenic to rice. It was determined that in strain ATCC 33617 the LuxR family quorum-sensing sensor/regulator TofR was inactive. Introducing the tofR gene of B. glumae AU6208 in strain ATCC 33617 restored its ability to produce toxoflavin and the LipA lipase. This study extends the role of AHL quorum sensing in rice pathogenicity through the regulation of a lipase which was demonstrated to be a virulence factor. It is the first report of a clinical B. glumae isolate retaining strong rice pathogenicity and finally determined that B. glumae can undergo phenotypic conversion through a spontaneous mutation in the tofR regulator.

A LuxR homologue of Xanthomonas oryzae pv. oryzae is required for optimal rice virulence.

SUMMARY: In Gram-negative bacteria a typical quorum sensing (QS) system usually involves the production and response to acylated homoserine lactones (AHLs). An AHL QS system is most commonly mediated by a LuxI family AHL synthase and a LuxR family AHL response regulator. This study reports for the first time the presence of a LuxR family-type regulator in Xanthomonas oryzae pv. oryzae (Xoo), which has been designated as OryR. The primary structure of OryR contains the typical signature domains of AHL QS LuxR family response regulators: an AHL-binding and a HTH DNA binding motif. The oryR gene is conserved among 26 Xoo strains and is also present in the genomes of close relatives X. campestris pv. campestris and X. axonopodis pv. citri. Disrupting oryR in three Xoo strains resulted in a significant reduction of rice virulence. The wild-type Xoo strains do not seem to produce AHLs and analysis of the Xoo sequenced genomes did not reveal the presence of a LuxI-family AHL synthase. The OryR protein was shown to be induced by macerated rice and affected the production of two secreted proteins: a cell-wall-degrading cellobiosidase and a 20-kDa protein of unknown function. By expressing and purifying OryR it was then observed that it was solubilized when grown in the presence of rice extract indicating that there could be a molecule(s) in rice which binds OryR. The role of OryR as a possible in planta induced LuxR family regulator is discussed.

Differential distribution of tocopherols and tocotrienols in photosynthetic and non-photosynthetic tissues.

Tocopherols and tocotrienols are vitamin E compounds, differing only in the saturation state of the isoprenoid side chain. Tocopherol biosynthesis, physiology and distribution have been studied in detail. Tocopherols have been found in many different plant species, and plant tissues. In contrast, comparatively little is known about the physiology and distribution of tocotrienols. These compounds appear to be considerably less widespread in the plant kingdom. In this study 80 different plant species were analysed for the presence of tocotrienols. Twenty-four species were found to contain significant amounts of tocotrienols. No taxonomic relation was apparent among the 16 dicotyledonous species that were found to contain tocotrienol. Monocotyledonous species (eight species) belonged either to the Poaceae (six species) or the Aracaceae (two species). A more detailed analysis of tocotrienol accumulation revealed the presence of tocotrienols in several non-photosynthetic tissues and organs, i.e. seeds, fruits and in latex, in concentrations up to 2000 ppm. No tocotrienols could be detected in mature photosynthetic tissues. However, we found the transient accumulation of low levels of tocotrienols in the young coleoptiles of plant species whose seeds contained tocotrienols. No measurable tocotrienol biosynthesis was apparent in coleoptiles, or in chloroplasts isolated from such coleoptiles. In line with these results, we found that tocotrienol accumulation in coleoptiles was not associated with chloroplasts. Based on our data, we conclude that tocotrienols may be transiently present in photosynthetically active tissues, however, it remains to be proven whether the tocotrienols are biosynthesised in such tissues, or imported from elsewhere in the plant.