Genome sequencing reveals the evolution and pathogenic mechanisms of the wheat sharp eyespot pathogen Rhizoctonia cerealis

The necrotrophic fungus Rhizoctonia cerealis is the causal agent of devastating diseases of cereal crops including wheat(Triticum aestivum).We present a high-quality genome assembly of R.cerealis Rc207,a virulent strain causing wheat sharp eyespot.The assembly(56.36 Mb)is composed of 17.87%repeat sequences and 14,433 predicted protein-encoding genes.The Rc207 genome encodes a large and diverse set of genes involved in pathogenicity,especially rich in those encoding secreted proteins,carbohydrateactive enzymes(CAZymes),peptidases,nucleases,cytochrome P450,and secondary metabolismassociated enzymes.Most secretory protein-encoding genes,including CAZymes,peroxygenases,dehydrogenases,and cytochrome P450,were up-regulated during fungal infection of wheat.We identified 831 candidate secretory effectors and validated the functions of 10 up-regulated candidate effector proteins.Of them,nine were confirmed as necrotrophic pathogen’s effectors promoting fungal infection.Abundant potential mobile or plastic genomic regions rich in repeat sequences suggest their roles in fungal adaption and virulence-associated genomic evolution.This study provides valuable resources for further comparative and functional genomics on important fungal pathogens,and provides essential tools for development of effective disease control strategies.

Changes in Activities of Antioxidant-Related Enzymes in Leaves of Resistant and Susceptible Wheat Inoculated with Rhizoctonia cerealis

In this study, the changes in activities of peroxidase （POD）, glutathione S-transferase （GST）, catalase （CAT）, and superoxide distmuase （SOD） in leaves of four wheat cultivars, including resistant cultivars Shannong 0431 and Shanhongmai, a mildsusceptible cultivar Lumai 21, and a susceptible cultivar Wenmai 6, were investigated to explore if these antioxidantrelated enzymes were involved in wheat defense response to Rhizoctonia cerealis attack. Results showed that the activities of these enzymes in different wheat cultivars varied greatly after challenging with R. cerealis. The POD activities in resistant cultivars Shannong 0431 and Shanhongmai increased much quicker after the inoculation and displayed much higher activity than that in susceptible cultivar Wenmai 6. In contrast, POD activity showed a slow accumulation and a delayed peak time. The activities of GST and CAT in Shannong 0431 and Lumai 21 increased obviously within 48 h post inoculation （hpi）, whereas SOD activity decreased dramatically within 24 hpi in Shannong 0431 and 24-48 hpi in Lumai 21. In susceptible cultivar Wenmai 6, the SOD and CAT activities didn’t change obviously, whereas the activity of GST was decreased. The activities of CAT and GST in Shanhongmai did not show significant difference compared with those in Wenmai 6. Results suggested that POD activity may be positively associated with wheat defense to R. cerealis.

The wheat receptor-like cytoplasmic kinase TaRLCK1B is required for host immune response to the necrotrophic pathogen Rhizoctonia cerealis

Receptor-like cytoplasmic kinases(RLCKs)represent a large family of proteins in plants.In Arabidopsis and rice,several RLCKs in subfamily VII(RLCKs-VII)have been implicated in pathogen-associated molecular pattern-triggered immunity and basal resistance against bacterial and fungal pathogens.However,little is known about roles of RLCKs-VII of the important crop common wheat(Triticum aestivum)in immune responses.Here,we isolated a RLCK-VII-encoding gene from wheat,designated as TaRLCK1B,and investigated its role in host immune response to infection of a necrotrophic fungus Rhizoctonia cerealis that is a major pathogen of sharp eyespot,a destructive disease of wheat.RNA-sequencing and RT-qPCR analyses showed that transcriptional level of TaRLCK1B was significantly higher in sharp eyespot-resistant wheat cultivars than in susceptible wheat cultivars.The gene transcription was rapidly and markedly elevated in the resistant wheat cultivars by R.cerealis infection.The TaRLCK1B protein was closely related to OsRLCK176,a rice resistance-related RLCKs-VII,with 84.03%identity.Virus-induced gene silencing plus wheat response to R.cerealis assay results indicated that silencing of TaRLCK1 impaired resistance to R.cerealis.Meantime,silencing of TaRLCK1 significantly elevated both the content of H2 O2(a major kind of reactive oxygen species,ROS)and the transcriptional level of the ROS-generating enzyme-encoding gene RBOH,but repressed the expression of the ROS-scavenging enzyme-encoding gene CAT1 at 18 hours after inoculation(hai)with R.cerealis.Taken together,these data suggested that TaRLCK1B was required for the early immune response of wheat to R.cerealis through modulating ROS signaling in wheat.

The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat

Wheat(Triticum aestivum L.)is an important staple crop for global human.The necrotrophic fungus Rhizoctonia cerealis is the causal pathogen of sharp eyespot,a devastating disease of wheat.Herein,we identified RcMEP1,a zinc metalloproteaseencoding gene from R.cerealis genomic sequences,and characterized its pathogenesis function.RcMEP1 expressed at markedly-high levels during R.cerealis infection process to wheat.The predicted protein RcMEP1 comprises of 287 amino acid residues and contains a signal peptide and a M43 metalloprotease domain harboring the active site motif(HEVGHWLGLYH).The assays of Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana leaves indicated that RcMEP1 is an apoplastic elicitor of cell death,and that the predicted signal peptide functions and is required for secretion and cell death-induction.The purified RcMEP1 protein and its M43 domain peptide were individually able to induce plant cell death and H2 O2 accumulation,and to inhibit expression of host chitinases when infiltrated into wheat and N.benthamiana leaves,while the M43 domain-deleting peptide and negative control lacked the capacity.Moreover,compared with the control pretreatment,the purified RcMEP1 protein or its M43-domain peptide resulted in enhanced pathogenesis in the inoculated wheat,whereas the M43 domain-deleting peptide failed.These results suggest that RcMEP1 acted as an important pathogenicity factor during R.cerealis infection to wheat and that its signal peptide and M43 domain are required for the secretion and pathogenesis of RcMEP1.This study provides insights into pathogenesis role of M43 domain-containing metalloproteases during R.cerealis infection to wheat.

Introgression of sharp eyespot resistance from Dasypyrum villosum chromosome 2VL into bread wheat

Wheat sharp eyespot, a stem disease caused by the soilborne fungus Rhizoctonia cerealis van der Hoeven,has become a threat to wheat production worldwide. Exploiting resistance resources from wild relatives of wheat is a promising strategy for controlling this disease. In this study, a new wheat–Dasypyrum villosum T2DS·2V#4L translocation line in the background of Chinese Spring(CS) showed stable resistance to R. cerealis. Introgression of the T2DS·2V#4L chromosome into wheat cultivar Aikang 58 by backcrossing produced a marked increase in sharp eyespot resistance in NIL-T2DS·2V#4L in comparison with NILT2DS·2DL, and no detrimental effects of 2V#4L on agronomic traits were observed in the BC2F2, BC2F2:3,and BC2F2:4generations. Flow-sorted sequencing of 2V#4L yielded 384.3 Mb of assembled sequence, and8836 genes were predicted of which 6154 had orthologs in at least one of the 2AL, 2BL, and 2DL arms of CS, whereas 1549 genes were unique to 2V#4L. About 100,000 SNPs were detected in genes of 2V#4L and2DL in 10 sequenced bread wheat cultivars. A Kompetitive Allele Specific Polymerase chain reaction and30 conserved ortholog sequence markers were developed to trace the 2V#4L chromatin in wheat backgrounds. T2DS·2V#4L compensating translocation lines represent novel germplasm with sharp eyespot resistance and the markers will allow rapid detection in breeding programs.

小麦纹枯病菌侵染过程的组织学研究

本文报道了小麦纹枯病菌（Rhizoctoniacerealis）侵染小麦的过程。病菌在穿透寄主之前产生侵染垫、菌丝圈以及形态简单的单附着胞等侵染结构。由侵染垫基部菌丝或附着胞产生的侵染菌丝直接或通过气孔侵入寄主，也可见菌丝直接侵入寄主；菌丝侵入寄主表皮后；迅速在受侵细胞内呈网状扩展，并直接穿透毗邻细胞壁，向其它细胞纵横扩展。受病组织出现细胞变形、变空；接近菌丝的质膜发生质壁分离，质膜断裂：叶绿体变形、变小或接近消失，类囊体被破坏，叶绿体内嗜饿颗料减少或无；线粒体解体等系列组织病变。

冬青抑菌活性的初步研究

采用生长速率法,测定冬青(Ilex purpurea Hassk)不同器官的提取液对小麦纹枯病菌(Rhizoctoniacerealis)、番茄褐斑病菌(Helminthosporiurn carposaprum)、番茄早疫病菌(Alternaria solani)、黄瓜褐斑病菌(Corynespora cassiicolai)的抑菌作用。结果表明,当浓度为0.1 g/mL时,冬青不同器官的提取液对4种病原菌菌丝生长都有一定的抑制作用,但不同部位提取液的抑菌活性不同,其中冬青果实和枝条提取液的抑菌活性较弱,树皮提取液的抑菌活性次之,叶片提取液的抑菌活性较强。

Composition of Wheat Rhizosphere Antagonistic Bacteria and Wheat Sharp Eyespot as Affected by Rice Straw Mulching

To assess the effect of rice straw mulching on changes of antagonistic bacteria and the incidence of wheat sharp eyespot, a multi-year field study was performed to compare unmulched plots and the plots mulched with rice straw for two or three years. Bacterial and fungal populations were evaluated in the cultures prepared from the wheat rhizosphere and bulk soils. Rice straw mulching increased the number of pseudomonas colony forming units in wheat rhizosphere and bulk soils. The proportion of total bacteria that were fluorescent pseudomonads was higher in mulched than in unmulched soil. Bacterial isolates antagonistic to Rhizoctonia cerealis were identified using an inhibition zone test. A series of these isolates were typed by partial sequencing of the 16S rRNA gene. Pseudomonads had higher antagonistic activity against R. cerealis than other species, and more than 80% of rhizosphere fluorescent pseudomonads were antagonistic to R. eerealis. The disease indices were lower in the mulched plots than in the unmulched control. These results suggest that rice straw mulching in a rice-wheat rotation increases the number of fluorescent pseudomonads. Additionally, these fluorescent pseudomonads may contribute to the control of wheat sharp eyespot.