Putative Phosphatase UvPsr1 Is Required for Mycelial Growth,Conidiation, Stress Response and Pathogenicity in Ustilaginonidea virens

Ustilaginoidea virens is the causal agent of rice false smut, which can be a highly destructive disease of rice. The plasma membrane phosphatase Psr1 proteins, which act as a regulator of the salinity stress response in yeast, are widely distributed across fungi, but their functional characterization is sketchy. In this study, we characterized the functions of Psr1 protein, UvPsr1, in U. virens. Analyses of the ΔUvpsr1 and its complementation strain showed that UvPsr1 is required for normal mycelial growth, conidiation and tolerance to oxidative, osmotic and cell wall stresses. When rice panicles were inoculated with the ΔUvpsr1 strains, no symptoms of false smut disease developed, showing that UvPSR1 also contributes to the pathogenicity of the fungus. The deletion mutant of UvPSR1 also appeared to produce a smaller titer of toxic compounds able to inhibit elongation of the germinated seeds. In conclusion, our results indicated that UvPsr1 is a new pathogenic factor of U. virens.     

UvSMEK1, a Suppressor of MEK Null,Regulates Pathogenicity,Conidiation and Conidial Germination in Rice False Smut Fungus Ustilaginoidea virens

Rice false smut, which is caused by Ustilaginoidea virens, is an emerging disease of rice spikelets in rice-growing areas worldwide. However, the infection mechanism of U. virens on rice spikelets is still unclear. Here, we characterized a suppressor of mitogen-activated protein kinase kinase or ERK kinase(MEK) null(UvSMEK1) in U. virens that is conserved among filamentous fungi. Compared with wild type U. virens strain P-1, Uv SMEK1 deletion mutants were defective in pathogenicity and conidial germination. In addition, conidiation of UvSMEK1 deletion mutants was significantly reduced on yeast extract tryptone(YT) plates, but increased in YT broth compared with the wild type. Compared with Uv SMEK1 expression level during the vegetative mycelia and conidiation stages, UvSMEK1 dramatically increased during infection of rice florets. Surprisingly, the Uv SMEK1 deletion mutants exhibited higher tolerance to H2 O2 and NaCl. In summary, presented evidence suggested that UvSMEK1 positively regulated pathogenicity, conidial germination and conidiation in YT broth, and negatively regulated conidiation on YT medium and tolerance to oxidative and osmotic stresses. The results enhance our understanding of the regulatory mechanism of pathogenicity of U. virens, and present a potential molecular target for blocking rice infection by U. virens.     

bZIP Transcription Factor UvATF21 Mediates Vegetative Growth,Conidiation, Stress Tolerance and Is Required for Full Virulence of Rice False Smut Fungus Ustilaginoidea virens

bZIP proteins are widely distributed in eukaryotic organisms and regulate a diverse range of physiological processes. Several bZIP proteins have previously been identified in Ustilaginoidea virens. However, the biological roles of these bZIP proteins in this pathogen are still unknown. Here, one of these bZIP protein coding genes, UvATF21, was functionally characterized. Targeted deletion of UvATF21 resulted in reduced conidiation and pathogenicity despite of the increased vegetative growth. The deletion mutants also significantly decreased the sensitivity to osmotic and oxidative stresses. Interestingly, deletion of UvATF21 exhibited different performances to cell wall integrity stress. These results indicated that UvATF21 played crucial roles in vegetative growth, conidiation, stress response, and full virulence in U. virens.     

稻曲病菌T-DNA插入突变体B2510的插入位点分析

以稻曲病菌T-DNA插入突变体库中致病力减弱突变菌株B2510为材料,通过分析T-DNA插入位点的侧翼序列和突变基因,分离出在稻曲病菌致病过程中起作用的基因。通过测定突变菌株B2510的生长速率、产孢能力及致病力发现,与野生型菌株P1相比,B2510田间接种表现为致病性减弱;在MM培养基上生长速率下降,而在PSA和TB3培养基中生长速率与野生型没有显著差异,但丧失产孢能力。Southern杂交显示T-DNA在突变菌株B2510中以双拷贝形式插入,利用TAIL-PCR技术扩增紧邻T-DNA两侧的侧翼序列,经过比对分析发现,T-DNA分别插在基因UV8b_1412的启动子区域和UV8b_1386的下游3′端,且稻曲菌基因组序列均未丢失,T-DNA上只有几个碱基发生变化。半定量RT-PCR分析基因的表达情况,显示两个基因在突变体B2510的表达量较P1均显著下降,推测T-DNA插入位点处的基因与稻曲病菌致病性相关,可能在某一阶段参与调控稻曲病菌在水稻上的致病过程。     

水稻稻曲病菌G蛋白β亚基基因的克隆、表达与序列分析

水稻稻曲病是由稻绿核真菌[Ustilaginoidea virens(Cooke)Tak]引起的一种常见的水稻后期穂部病害。G蛋白可能参与了稻曲病菌的致病过程。为了研究G蛋白在病菌致病过程中的作用,分离并分析了稻曲菌的G蛋白β亚基编码基因。根据丝状真菌G蛋白β亚基编码基因的同源保守序列设计简并引物,采用同源克隆和热不对称交错PCR的方法,分离得到了稻曲菌的G蛋白β亚基全编码基因序列。该序列的长度为2037bp,包含4个内含子,5个外显子和1个编码359个氨基酸的开放阅读框。根据克隆到的UvGβ1设计引物,通过RT-PCR克隆到包含整个开放阅读框的cDNA序列。该基因的DNA序列和cDNA序列在GenBank中注册的登记号分别为GU014921和GU065745。系统进化分析表明该片段与栗疫病菌(Cryphonectria parasitica)的G蛋白β亚基基因亲缘关系最近。将该基因的整个开放阅读框连接于pET-30a构建原核表达载体,通过诱导获得了重组蛋白。     

稻曲病菌UvHog1基因的克隆及表达分析

稻曲病由稻曲病菌引起,是水稻穗部的重要病害。利用同源克隆和RACE技术,根据丝状真菌相对保守的氨基酸序列设计简并引物,从稻曲病菌中分离了丝裂原活化蛋白激酶(MAPK)编码基因的全长cDNA序列,命名为UvHog1。UvHog1含有MAPK保守的蛋白激酶激活域(TGY),序列与稻瘟病菌MgOsm1(AAF09475.1)、球孢白僵菌BbHog1(AAS77871.1)、烟曲霉AfOsm1(XP752664.1)和隐球酵母CrHog1(AAM26267.1)等MAPK高度同源,相似性分别为95%、93%、88%和81%。系统聚类结果表明,UvHog1与酵母Hog1MAPK同源。在盐胁迫条件下,UvHog1的相对表达量明显降低,表明UvHog1参与了对盐胁迫的信号响应。     

Integrated Approach to Control False Smut in Hybrid Rice in Sichuan Province,China

Severe epidemic of false smut, caused by Ustilaginoidea virens(Cooke) Takahashi(teleomorph Villosiclava virens) has been reported in different parts of Asia and America. Different fungicides or bio-control agents against false smut were applied at different times before heading on a susceptible rice variety Pu-6. A control efficiency as high as 91.92% was resulted from spraying 2.5% Wenquning, a suspension of Bacillus subtilis in solution of validamycin with 4.5 L/hm2 at 6 d before heading. Among the 186 hybrid rice screened in 2010, low significant correlations between the dates of full heading, rates of infected plants and panicles as well as the number of infected florets were found, with the correlation coefficients ranging from 0.2331 to 0.5212. However, significant difference in susceptibility coefficients was also found between the varieties which had the same dates of full heading. In the plot experiments to compare the susceptibility in 2011, the average rates of infected panicles of Yixiangyou 2168, Chuanxiangyou 3, Dexiang 4103, Yixiangyou 2115, Nei5 you 317, Yangxinyou 1 were significantly lower than those of the control varieties Gangyou 725 and Gangyou 188 at the disease nursery located at Qionglai, Sichuan Province, China. When Neixiangyou 8156 and Nei5 you 317 were sprayed with 2.5% Wenquning at 4.5 L/hm2 for two times at 6 d before and 1 d after heading, respectively, the control efficiencies of Nei5 you 317 and Neixiangyou 8156 were respectively 100% and 82.24% compared to that of Gangyou 725. Satisfactory control effects had also obtained by single spray of 2.5% Wenquning at 4.5 L/hm2 at 5–6 d before heading. Therefore, less susceptible hybrid rice in combination with spraying Wenquning at 5–6 d before heading was suggested for the disease control in Sichuan Province, China.     

Rice Aroma: A Natural Gift Comes with Price and the Way Forward

Aromatic rice belongs to a small but important sub-group of rice, which is highly regarded for its excellent aroma and superior grain quality. Aromatic rice, especially Basmati-and Jasmine-type rice, is being traded at a high price in the local and global markets. Genetically, rice aroma is a phenotypical expression of spontaneous recessive mutations of the OsBadh2 gene(also known as fgr/badh2/osbadh2/os2AP gene). These mutations inhibit the flow of γ-aminobutyraldehyde(GAB-ald) to γ-aminobutyric acid(GABA), and consequently, the accumulated GAB-ald is diverted to a potent flavour component 2-acetyl-1-pyrroline(2AP) by a non-enzymatic reaction with methylglyoxal. The natural incidence of non-functional osbadh2 mutation along with selection and nursing by the farmer from the ancient time makes rice aroma as a prominent natural gift. As GABA and methylglyoxal play significant roles in stress tolerance, and their biosynthesis is strictly regulated in rice plants, the accumulation of 2AP in aromatic rice depends on the interaction of various genetic and environmental factors, and its production may come at some costs of sacrificing tolerance. This review focused on some potential underlying genes in the 2AP and GABA biosynthesis pathways, and analyzed most aspects of aroma formation in rice, and summarized the molecular mechanism of aroma production together with its genetic and non-genetic influencing factors. The present review also stated approaches to produce high-quality aromatic rice via developing novel cultivars and with good agronomic knowledge-based practice.     

Impacts of biocontrol products on Rhizoctonia disease of potato and soil microbial communities,and their persistence in soil

Four commercial biocontrol formulations (Bacillus subtilis GB03, Burkholderia ambifaria type Wisconsin isolate J82, Trichoderma virens Gl-21, and Trichoderma harzianum strain T-22), a chemical seed treatment (thiophanate-methyl, mancozeb, and cymoxanil mixture, TMC), and a combination chemical/biological treatment, were compared with no-pathogen and pathogen-treated controls, and monitored in two field seasons in Maine for their effects on the development of Rhizoctonia disease of potato and soil microbial community characteristics. All treatments reduced the incidence and severity of stem canker (37–75% reduction) relative to the pathogen control over both years, with the best control provided by B. subtilis and the combination chemical/biological treatment (TMC/Bamb). Both bacterial treatments (B. subtilis and Bu. ambifaria) reduced severity of black scurf in both years, and T. virens reduced scurf in one year, with reductions of 11–20% relative to the pathogen control. Over both years, the B. subtilis, T. virens, and TMC/Bamb treatments increased total and marketable yield, and Bu. ambifaria increased marketable yield, by 11–15% relative to the pathogen control. Substantial populations of the added fungal agents, but not the bacteria, were detected in bulk soil at the end of the growing season. Biocontrol treatments also significantly (P < 0.05) affected soil microbial community characteristics, as assessed by single carbon source substrate utilization (SU) and whole soil fatty acid methyl ester (FAME) profiles. Bacterial biocontrol treatments generally resulted in higher microbial activity and substrate utilization. Some effects on soil microbial communities were also observed the following spring (1 yr after application). This research indicates that biocontrol treatments can assist in the control of Rhizoctonia disease of potato, persist in soil to some degree, and have significant effects on soil microbial communities long after application.     

Effect of Infection by Beauveria bassiana and Metarhizium anisopliae on the Feeding of Uvarovistia zebra

To identify the susceptibility of long-horned grasshoppers to entomopathogenic fungi, the effect of infection with the fungi Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) on food consumption by Uvarovistia zebra (Uvarov) (Orthoptera: Tettigoniidae) was investigated. Preliminary results showed that both fungi had a negative effect on food consumption of the insects. For both fungi a significant reduction of food consumption and faeces production by insects were observed between the highest spore concentration (5 × 10⁶ spores/ml) and other treatments. Compared with control insects, the insects treated with 5 × 10⁶ spores/ml of B. bassiana and M. anisopliae showed 60 and 63% reduction in mean food consumption/insect, respectively. The corrected cumulative percent mortality of the insects treated with the highest concentration of B. bassiana and M. anisopliae were 57.7 and 55.5%, respectively. This was the first account of these entomopathogenic fungi being used against a species from this family, therefore based on the results obtained from this research, it could be said that the fungi have pathogenicity effect on U. zebra as a long-horned grasshopper.     

Genetic analysis of rainfed lowland rice drought tolerance under naturally-occurring stress in eastern India: Heritability and QTL effects

Drought tolerance is an important rainfed rice breeding objective, but because the heritability (H) of yield under drought stress is thought to be low, secondary physiological traits are considered better targets for selection than yield under stress per se. This assumption has rarely been tested, and there are no reports on H for yield under drought stress from experiments repeated over seasons in rainfed lowland rice. To assess the potential for improving yield under drought stress via direct selection, and to identify associated quantitative trait loci (QTL), doubled haploid lines with a narrow range of flowering dates, derived from the population CT9993-5-10-1-M/IR62266-42-6-2, were screened under full irrigation and severe drought stress induced by draining the paddy before flowering in 2000–2002 at Raipur, India. Drought stress reduced mean yield by 80%. H was similar in stress and non-stress trials, as was the relative magnitude of the genotype and genotype × year variances. The genetic correlation between yield in stress and non-stress conditions was 0.8, indicating that about 64% of the genetic variation for yield under stress was accounted for by differences in yield potential also expressed in irrigated environments. These results indicate that direct selection for yield under drought stress can produce yield gains under stress without reducing yield potential. There was no secondary trait for which selection resulted in greater predicted response in yield under stress than direct selection for stress yield per se. A QTL was detected on chromosome 1 near sd1 that explained 32% of the genetic variation for yield under stress, but only 4% under non-stress. Its effect was consistent across years. This QTL accounted for much of the variation in drought yield not accounted for by variation in yield potential.     

Ustilaginoidea virens: A Fungus Infects Rice Flower and Threats World Rice Production

Rice false smut disease, which is caused by the fungus Ustilaginoidea virens, is currently one of the most devastating rice fungal diseases in the world. Rice false smut disease not only causes severe yield loss and grain quality reduction, but also threatens food safety due to its production of mycotoxins. In this review, the most recent progresses regarding the life cycle, infection processes, genome and genetic diversity, pathogenic gene and disease resistance in rice were summarized in order to provide theoretical basis for the control of U. virens. We also proposed some future directions and key questions that need to be addressed for a better understanding of the molecular mechanism that leads to rice false smut disease and the prospects for sustainable control of rice false smut.