Molecular characterization of the avrXa7 locus from Xanthomonas oryzae pv. oryzae field isolates

The effector gene avrXa7 from Xanthomonas oryzae pv. oryzae has avirulence function in rice with the Xa7 resistance gene and confers pathogenic fitness (aggressiveness). Field strains of X. oryzae pv. oryzae displayed a diversity of phenotypes on rice ranging from complete to partial loss of these functions. To understand the molecular basis for variation in avrXa7 function, we sequenced the alleles from seven field strains. The avrXa7 gene is an avrBs3/pthA family member and contains nuclear localization signal sequences and an acidic activation domain (AAD) in the 3′ end and a central region containing repeated sequences, all of which are important for avirulence and aggressiveness. Sequence analysis revealed changes in the avrXa7 alleles ranging from a point mutation to multiple mutations spread throughout the alleles. Some strains with identical mutant alleles exhibited different levels of aggressiveness to rice, suggesting the presence of second mutations. A point mutation found at the beginning of the AAD of one avrXa7 allele was reconstructed in the wild type gene; this mutant exhibited partial loss of avirulence and aggressiveness. Our data suggest that adaptation of X. oryzae pv. oryzae to Xa7 rice fields involves not only alterations in avrXa7, but may include changes in other gene family members resulting from recombination between family members.     

Regulatory network of hrp gene expression in Xanthomonas oryzae pv. oryzae

Like other plant-pathogenic bacteria, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, has hrp genes that are indispensable for its virulence. The hrp genes are involved in the construction of the type III secretion (T3S) apparatus, through which dozens of virulence-related proteins, called effectors, are directly secreted into plant cells to suppress and disturb plant immune systems and/or induce plant susceptibility genes. The expression of hrp genes is strictly regulated and induced only in plants and in certain nutrient-poor media. Two proteins, HrpG and HrpX, are known as key regulators for hrp gene expression. Great efforts by many researchers have revealed unexpectedly that, besides HrpG and HrpX, many regulators are involved in this regulation, some of which also regulate the expression of virulence-related genes other than hrp. Moreover, it has been found that HrpG and HrpX regulate not only hrp genes and effector genes but also genes unrelated to the T3S system. These findings suggest that the expression of the hrp gene is orchestrally regulated with other virulence-related genes by a complicated, sophisticated regulatory network in X. oryzae pv. oryzae.     

Involvement of OsNPR1/NH1 in rice basal resistance to blast fungus Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a major threat to worldwide rice production. Plant basal resistance is activated by virulent pathogens in susceptible host plants. OsNPR1/NH1, a rice homolog of NPR1 that is the key regulator of systemic acquired resistance in Arabidopsis thaliana, was shown to be involved in the resistance of rice to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae and benzothiadiazole (BTH)-induced blast resistance. However, the role of OsNPR1/NH1 in rice basal resistance to blast fungus M. oryzae remains uncertain. In this study, the OsNPR1 gene was isolated and identified from rice cultivar Gui99. Transgenic Gui99 rice plants harbouring OsNPR1-RNAi were generated, and the OsNPR1-RNAi plants were significantly more susceptible to M. oryzae infection. Northern hybridization analysis showed that the expression of pathogenesis-related (PR) genes, such as PR-1a, PBZ1, CHI, GLU, and PAL, was significantly suppressed in the OsNPR1-RNAi plants. Consistently, overexpression of OsNPR1 in rice cultivars Gui99 and TP309 conferred significantly enhanced resistance to M. oryzae and increased expression of the above-mentioned PR genes. These results revealed that OsNPR1 is involved in rice basal resistance to the blast pathogen M. oryzae, thus providing new insights into the role of OsNPR1 in rice disease resistance.     

Pathogenesis-related gene expression in rice is correlated with developmentally controlled Xa21-mediated resistance against Xanthomonas oryzae pv. oryzae

Disease resistance mediated by the resistance gene Xa21 is developmentally controlled in rice. We examined the relationship between Pathogenesis Related (PR) defense gene expression and Xa21-mediated developmental disease resistance induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1a, OsPR1b, and OsPR1c genes were cloned and their induction was analyzed, in addition to the OsPR10a gene, at the juvenile and adult stages in response to a wildtype Xoo strain that induces a resistance response (incompatible interaction) and an isogenic mutant Xoo strain that does not (compatible interaction). We found that the adult stage leaves are more competent to express these OsPR1 genes and that the Xa21 locus is required for the highest levels of induction.     

Characterization of interactions between barley and various host-specific subgroups of Magnaporthe oryzae and M. grisea

The Magnaporthe oryzae–M. grisea species complex is composed of several host-specific subgroups, but does not contain a barley-specific subgroup. To characterize the relationship between barley and these subgroups, we inoculated 24 barley cultivars separately with each of 18 isolates from various hosts. The interactions between these cultivars and isolates included various reactions from nonhost-like immune responses to typical host responses. Evenly closely related isolates of the blast fungi caused such contrasting reactions. The immune responses of barley cultivars against a Setaria isolate, Si-1J, were examined in detail. An infection assay with near-isogenic fungal strains suggested that PWT1, which was first identified as a major gene conditioning the avirulence of Si-1J on wheat, was involved in the avirulence on two-thirds of the barley cultivars. At the cytological level, the immune responses were associated with both papilla formation and hypersensitive reaction (HR). Of these two, however, HR played a more critical role than papilla formation. Studying the interactions of barley with M. oryzae and M. grisea may reveal various steps in the process of host specialization of a parasite species and the concomitant evolution of host resistance.     

Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae

To investigate mitotic homologous recombination (HR) in Pyricularia oryzae, we created an HR detection system. The system consists of two non-functional enhanced yellow fluorescent protein (YFP) and blasticidin S deaminase (BSD) fusion genes (YFP::BSD). If mitotic HR occurs between the two non-functional genes in the genome, restoration of the functional YFP::BSD gene can be expected. The expression of the functional YFP::BSD gene can be detected by both YFP fluorescence and resistance against BS. When the P. oryzae genome was transformed simultaneously with two non-functional genes, all six lines of transformants with both genes had some portion of their hyphae exhibiting YFP fluorescence and BS resistance during growth. Up to ca. 10 % of conidia harvested from the mycelium of each of the six lines had YFP fluorescence, suggesting that HR consistently occurs during mycelium growth. To determine whether and how the HR-mediated phenotypic changes occurred at the DNA level, we analyzed the genomic DNA of BS-resistant mycelia by PCR-RFLP and sequencing and were able to confirm the existence of a restored functional YFP::BSD gene and a non-functional recombinant gene. Collectively, these findings demonstrate that we established a successful HR detection system for P. oryzae, which can be used for other plant pathogens, and that mitotic HR actually occurs in P. oryzae and constitute the first experimental evidence for mitotic HR in a fungus.     

Functional analysis and expressional regulation of wxoE and wxoF in lipopolysaccharide (lps) biosynthesis gene cluster I of Xanthomonas oryzae pv. oryzae

wxoE and wxoF, two genes in the lipopolysaccharide (LPS) biosynthesis cluster I of Xanthomonas oryzae pv. oryzae (Xoo) that have not been characterized, were mutated by transposon insertion. Transposon mutants of wxoE and wxoF were nonpathogenic to rice. In LPS analysis on SDS-PAGE, Low mobility bands regarded as LPS O-antigen complex were observed in wild-type strain KACC10859 and mutant wxoD, but not in LPS profiles of wxoA, wxoB, wxoC, wxoE and wxoF mutants. In addition, exopolysaccharide (EPS) production from wxoE and wxoF mutant strains were dramatically reduced. WxoE protein showed enzymatic activity resembling that of cystathionine γ-lyase and specificity to cystathionine substrates. WxoF showed significant homology with methyltransferases that may function in the methylation of sugars in LPS biochemical modifications. Western blot analysis demonstrated WxoF is located in membrane and the lps genes involving wxoE and wxoF in cluster I are cotranslated in an operon that is dependent on a promoter with a polar fashion.     

Infection by Magnaporthe oryzae chrysovirus 1 strain A triggers reduced virulence and pathogenic race conversion of its host fungus, <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is associated with an impaired growth phenotype of its host fungus, Magnaporthe oryzae. In this report, we assayed the virulence and pathogenicity of MoCV1-A-infected and MoCV1-A-free M. oryzae on rice plants. MoCV1-A infection did not affect virulence-associated fungal traits, such as conidial germination and appressorium formation. However, after punch inoculation of leaves on rice plants, MoCV1-A-infected strain formed smaller lesions than the MoCV1-A-free strain did on all rice varieties tested, showing that MoCV1-A infection resulted in reduced virulence of host fungi in rice plants. In contrast, after spray inoculation of rice seedlings, in some cases, MoCV1-A-infected and MoCV1-A-free strains caused different lesion types (resistance to susceptible, or vice versa) on individual international differential rice varieties. However, we did not find any gain/loss of the fungal avirulence genes by PCR, suggesting that MoCV1-A infection can convert the pathogenicity of the host M. oryzae from avirulence to virulence, or from virulence to avirulence, depending on the rice variety. We also confirmed the correlation of these race conversion events and invasive hyphae growth of the fungi in a leaf sheath inoculation assay. These data suggested that MoCV1-A infection generally confers hypovirulence to the fungal host and could be a driving force to generate physiological diversity, including pathogenic races.     

Impact of tricyclazole and azoxystrobin on growth,sporulation and secondary infection of the rice blast fungus,Magnaporthe oryzae

BACKGROUND: Rice blast, caused by Magnaporthe oryzae B. Couch sp. nov., is one of the most destructive rice diseases worldwide, causing substantial yield losses every year. In Italy, its management is based mainly on the use of two fungicides, azoxystrobin and tricyclazole, that restrain the disease progress. The aim of this study was to investigate and compare the inhibitory effects of the two fungicides on the growth, sporulation and secondary infection of M. oryzae. RESULTS: Magnaporthe oryzae mycelium growth was inhibited at low concentrations of azoxystrobin and relatively high concentrations of tricyclazole, while sporulation was more sensitive to both fungicides and was affected at similarly low doses. Furthermore, infection efficiency of conidia obtained from mycelia exposed to tricyclazole was affected to a higher extent than for conidia produced on azoxystrobin‐amended media, even though germination of such conidia was reduced after azoxystrobin treatment. CONCLUSIONS: This study presents for the first time detailed azoxystrobin and tricyclazole growth–response curves for M. oryzae mycelium growth and sporulation. Furthermore, high efficacy of tricyclazole towards inhibition of sporulation and secondary infection indicates an additional possible mode of action of this fungicide that is different from inhibition of melanin biosynthesis. Copyright © 2012 Society of Chemical Industry     

Phosphohexose mutase of Xanthomonas oryzae pv. oryzicola is negatively regulated by HrpG and HrpX,and required for the full virulence in rice

The genome of Xanthomonas oryzae pv. oryzicola annotates one uncharacterized gene, XOC_3841, only one ORF in this strain is annotated to encode Phosphohexose mutase (XanA), which reversibly converts glucose 1-phosphate to glucose 6-phosphate that implicates in the carbon metabolism pathways. However, it is unclear whether the XanA-coding gene is involved in the full virulence of X. oryzae pv. oryzicola. In this report, we showed that the mutagenesis in unique xanA, led the pathogen effectively to unable to utilize glucose and galactose for growth. The expression of xanA was strongly induced by glucose, sucrose, fructose, mannose or galactose at least 3 times higher than that by non-sugar NY medium. Intriguingly, xanA promoter region contains an imperfect PIP-box (plant-inducible promoter) (TTCGC-N16-TTCGA), and the expression of xanA was inducible in rice suspension cells rather than in a nutrient-rich (NB) medium and negatively regulated by a key hrp regulatory HrpG and HrpX cascade. More importantly, mutation in xanA resulted in impairment of bacterial growth and virulence in planta, and reduced bacterial cell motility and extracellular polysaccharides (EPS) production in media. In addition, the lost properties mentioned above in RΔxanA were completely restored to the wild-type level by the presence of xanA in trans. All these results suggest that xanA is required for EPS production, cell motility and the full virulence of X. oryzae pv. oryzicola.     

Overwintering of <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> in wild infected foxtails

The rice blast fungus Pyricularia oryzae mainly overwinters in infested rice organs stored indoors, whereas it is difficult or impossible for the pathogen to overwinter outdoors. By contrast, blast pathogens infecting weed grasses must overwinter outdoors every winter to continue their life cycle. In this study, we investigated the overwintering location of P. oryzae infecting wild, green, and giant foxtails to identify the mechanism that enables them to overwinter. Recovery of P. oryzae was tested in seeds of wild foxtail collected from the soil surface from December to April over three winters. No P. oryzae was recovered from the seed samples of any wild foxtail collected at the ends of the three experimental periods in April. Recovery was also tested from blast lesions on leaves and seeds sampled from withered green foxtail in the experimental field of Saga University from November to April during two winters. In contrast to seeds on the soil surface, P. oryzae survived in lesions and seeds at the ends of the two experimental periods during April, suggesting that withered host plants could be the overwintering site of the pathogen. Rice plants are reaped and removed from paddy fields after harvesting. Thus, withered, standing plants may be available solely to blast pathogens infecting wild grasses, possibly explaining the higher winter survival frequency of weed pathogens than that of rice blast pathogens outdoors.     

Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

A single amino acid substitution in the SdhB protein of succinate dehydrogenase determines resistance to amicarthiazol in Xanthomonas oryzae pv. oryzae

BACKGROUND: Xanthomonas oryzae pv. Oryzae Ishiyama, a causal agent of rice bacterial leaf blight, was found to be sensitive in vitro to the systemic fungicide amicarthiazol (2‐amino‐4‐methylthiazole ‐5‐carboxanilide), which is a potent inhibitor of succinate dehydrogenase (SDH, EC 1.3.99.1). This paper aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzae to amicarthiazol. RESULTS: UV‐induced resistant mutants of X. oryzae pv. oryzae to amicarthiazol were isolated. The activity of SDH in wild‐type X. oryzae pv. oryzae was strongly inhibited by amicarthiazol, while that in resistant mutants was insensitive, although their SDH activity was decreased compared with the wild‐type sensitive strain without amicarthiazol. A mutation of Histidine₂₂₉ (CAC) to Tyrosine₂₂₉ (TAC) was identified in sdhB, which encoded the iron–sulfur protein subunit of SDH. The sdhB from the mutant was ligated into a cosmid, pUFR034, to generate pUFR034RAX, which conferred resistance to amicarthiazol when transformed into the wild‐type sensitive strain. CONCLUSION: A mutation of His₂₂₉ (CAC) to Tyr₂₂₉ (TAC) in SdhB was responsible for determining amicarthiazol resistance. Copyright © 2010 Society of Chemical Industry     

DNA typing and virulence determination of Xanthomonas oryzae pv. oryzae population for the management of bacterial leaf blight of rice in Udham Singh Nagar,India

Bacterial leaf blight caused (BLB) by Xanthomonas oryzae pv. oryzae (Xoo) is the most serious biotic constraint of rice in Udham Singh Nagar (U.S. Nagar), India. We have experienced severe BLB outbreaks, causing total crop failure during 2007–2010. The severity of losses incurred due to BLB in U.S. Nagar necessitates the development of an ecology-conscious, effective and durable management strategy. Our interests here were focused to obtain information on the haplotypic and pathogenic variability of Xoo to develop effective breeding strategy for BLB resistance. Our results indicate that the currently existing population of Xoo in U.S. Nagar is highly virulent and genetically diverse. Twenty one haplotypes were detected among a collection of 193 strains by each of the PCR technique used i.e. rep-PCR and IS-PCR. The high value of total haplotypic diversity (H_T ⁼0.79) reflected the genetic heterogeneity of Xoo population infecting popular varieties. Strains responsible for severe BLB outbreaks were grouped into eight distinct lineages with significant bootstrap values (p?>?75). Virulence assay revealed the presence of 11 pathotypes. The interaction of lines with strains was found significant (p?<?0.01) confirming the pathogenic specialization of Xoo in U.S. Nagar. Most of the single major resistance genes used in rice breeding program at International Rice research Institute (IRRI) was defeated by Xoo strains, at the same time; effectiveness of Xa21 to all the pathotypes is encouraging. The potential of Xa21 alone or in combination with xa13 and xa5 could be exploited for pyramiding into well-adapted rice cultivars for the effective management of the pathogen in this region. This is the first report on the population structure of Xoo in U.S. Nagar, India.     

Effect of fumonisin B<Subscript>1</Subscript> on the emergence,growth and ceramide synthase gene expression of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp)

Rice production is currently expanding from the south-eastern regions of Australia into northern Australia where indigenous species of wild rice occur widely. A survey of fungal diseases on wild (Oryza australiensis, Oryza spp.) and cultivated rice (Oryza sativa) in North Queensland, Australia, in May 2014 revealed a diverse range of fungal genera species, including important pathogens of cultivated rice. Whilst a single isolate of Magnaporthe oryzae (causal agent of rice blast) was obtained from wild rice, Bipolaris oryzae (causal agent of brown spot) was the predominant pathogen detected under North Queensland conditions. For the first time for Australia, we report Rhizoctonia oryzae-sativae (causal agent of aggregate sheath spot) occurring on wild rice. Other pathogens detected on wild rice included Curvularia lunata, Cochliobolus intermedius, Cochliobolus geniculatus, and Fusarium equiseti present in the majority of wild rice samples. Nearby cultivated rice fields harboured additional pathogens not found in wild rice including Fusarium graminearum, Leptosphaeria spegazzinii and Cochliobolus lunatus, causing scab disease, glume blight and leaf blight, respectively. We also confirmed that Bipolaris oryzae from wild rice can infect cultivated rice. This study highlights the importance of wild rice species as alternative hosts harbouring pathogens of cultivated rice and the likely disease threats to expansion of cultivated rice into the same region(s) where wild rice is endemic.     

Management of rice blast (Pyricularia oryzae): implications of alternative hosts

Rice blast (Pyricularia oryzae) has become a serious disease on commercial rice (Oryza sativa) crops in northern Australia and is present there on wild rice (O. australiensis). Characterisation of the host range of P. oryzae is fundamental to both reducing disease spread and to preventing development of epidemics via better management of Poaceae inoculum reservoirs in Australia. Studies on response of three different wild O. australiensis sources toward four isolates of P. oryzae showed all genotypes very susceptible to three isolates [WAC13466 (race IA-1), BRIP53376 (race IB-3), NT2014a (race unknown)], but resistant to isolate BRIP39772 (race IA-3). Studies to investigate levels of blast disease development following inoculation on a range of Poaceae hosts showed both P. oryzae isolates (WAC13466, BRIP53376) were highly virulent on barley (disease index, DI?=?100%), and on Phalaris and O. australiensis (DI?=?70%). However, isolate BRIP53376 showed a significantly higher level of aggressiveness (DI ~80%) on ryegrass, wild oat and rice. Of the two wheat cultivars tested, only one cultivar showed disease and only from WAC13466 (DI ~30%). Sweet corn and goosegrass were also susceptible to both blast isolates, but DI was <50%. That P. oryzae was virulent across these diverse Poaceae hosts, highlights, for Australia, the possibility for these species in, first, harbouring P. oryzae isolates highly virulent to commercial rice, second, fostering spread of rice-attacking P. oryzae strains into regions currently free of rice blast, and third, potential for these alternative host species to encourage development of more virulent host-specific strains of P. oryzae. The current study is an important step towards facilitating improved crop protection in the medium and long term from reducing P. oryzae disease epidemics via a better understanding and management of inoculum reservoirs in Australia.

     

Classification and parasitic specialization of blast fungi

Pyricularia oryzae (Magnaporthe oryzae), a causal agent of blast diseases on staple gramineous crops, is a model organism listed as the most important economically and scientifically of the top 10 fungal pathogens by fungal molecular pathologists. Although we are now in an era of genome-enabled analysis, we need to understand the history of the pathogen’s taxonomy, classification, and parasitic specialization in addition to recent research advances. In this review, we focus on these rather fundamental topics. First, the history of classification, including the discovery of its sexual stage and designation, is overviewed. Based on recent results of phylogenetic analysis of Magnaporthaceae isolates, blast fungi are suggested to constitute a robust population that is not congeneric with Magnaporthe salvinii, the type species of Magnaporthe. Second, genetic mechanisms involved in its parasitic specialization into host-specific subgroups and races are outlined. Implications of recent molecular data for resistance breeding are discussed.     

Effect of tricyclazole on growth and secondary metabolism in Pyricularia oryzae

Tricyclazole [5-methyl-1,2,4-triazolo (3,4-b)-benzothiazole] controls rice blast disease caused by Pyricularia oryzae at concentrations (5–10 μg/ml) which do not inhibit growth of the pathogen in vitro. However, concentrations of 1 μg/ml or less inhibit melanin formation in the fungus. Production of pyriculol by the pathogen is usually enhanced by 10 μg/ml of tricyclazole, whereas production of 3,4-dihydro-4,8-dihydroxy-1(2H)-naphthalenone is strongly inhibited or markedly reduced and delayed. Evidence suggests that tricyclazole blocks aspects of polyketide metabolism in P. oryzae which may have a role in pathogenicity.     

Disease Note: Identification of Curvularia oryzae as cause of leaf spot disease on oil palm seedlings in nurseries of Thailand

The causal agent of leaf spot disease of oil palm (Elaeis guineensis) seedling nurseries in Thailand was identified as Curvularia oryzae. The fungus was isolated from leaves with disease symptoms, characterized by morphological properties, and pathogenicity tested. The identity of the phytopathogenic fungus was confirmed through polymerase chain reaction analysis using internal transcribed spacer (ITS) primers, which amplified about a 1 kb product. Sequencing this DNA product confirmed this pathogen was C. oryzae. Furthermore, the pathogenicity test showed that C. oryzae could infect oil palm seedlings.