Rapid detection of the rice false smut fungus Ustilaginoidea virens by lateral flow strip-based recombinase polymerase amplification assay

Rice false smut,caused by Ustilaginoidea virens,is a devastating disease that greatly reduces rice yield and quality.However,controlling rice false smut disease is challenging due to the unique infection mode of U.virens.Therefore,there is a need for early diagnosis and monitoring techniques to prevent the spread of this disease.Lateral flow strip-based recombinase polymerase amplification(LF-RPA)overcomes the limitations of current U.virens detection technologies,which are time-consuming,require delicate equipment,and have a high false-positive rate.In this study,we used a comparative genomics approach to identify Uv_3611,a specific gene of U.virens,as the target for the LF-RPA assay.The designed primers and probe efffectively detected the genomic DNA(gDNA)of U.virens and demonstrated no cross-reactivity with related pathogens.Under optimal conditions,the LF-RPA assay demonstrated a sensitivity of 10 pg of U.virens gDNA.Additionally,by incorporating a simplified PEG-NaOH method for plant DNA extraction,the LF-RPA assay enabled the detection of U.virens in rice spikelets within 30 min,without the need for specialized equipment.Furthermore,the LF-RPA assay successfully detected U.virens in naturally infected rice and seed samples in the field.Therefore,the LF-RPA assay is sensitive,efficient,and convenient,and could be developed as a kit for monitoring rice false smut disease in the field.

Analysis on Quantitative Trait Loci for Resistance to Rice False Smut under Different Environmental Conditions

A recombinant inbred line （RILs） population with 157 lines derived from an inter-subspecies cross of Daguandao （japonica）/IR28 （indica） by the single seed de-scent method was used to detect quantitative trait loci （QTLs） conferring resistance to rice false smut caused by Ustilaginoidea virens（Cooke） Takahashi in Nanjing and Yangzhou. The disease rate index of the two parents and 157 RILs caused by rice false smut were scored and the QTLs for rice false smut resistance were detected accordingly by QTL Cartographer software. Eight QTLs control ing false smut resis-tance were detected on chromosomes 1, 2, 4, 8, 10, 11 and 12, respectively, with the phenotypic variance of 8.6%-22.5%. There were five QTLs detected in Nanjing and Yangzhou, respectively, and only two QTLs were found in both two years, the phenotypic variation was explained by individual QTL ranged from 18.0% to 18.9% for these two QTLs, and the additive effects of these two QTLs contributed to the 8.0%-14.6% decrease of disease index and therefore the disease resistance increased. The direction of the additive effects at six loci qFsr1, qFsr2, qFsr8, qFsr10a, qFsr11 and qFsr12 coincided with that predicted by phenotypes of the parents, and the IR28 al eles at these loci had positive effect against rice false smut while the negative effects were found in Daguandao al eles at qFsr4 and qFsr10b. Both qFsr10a and qFsr11 should be useful in rice breeding for resistance to rice false smut in marker-assisted selection （MAS） program.

Progress in the Study of False Smut Disease in Rice

Rice false smut disease is an increasing concern for research and production, not only because of the increasing epidemic occurrence in rice production, but also the intriguing specific pathogenesis of the disease to be a unique pathological system to enrich the molecular mechanism of plant-microbe interaction. Progresses have been achieved in the pathogen phylogenetic placement, the alternative hosts, the pathogen morphology and diversity, the toxins generated by false smut balls, the artificial inoculation method, and the pathogen transformation as well as rice resistance to the disease. However, it is still controversy on the infection process. It is not clear how the life cycle of this pathogen is coupled with the disease cycle. This review summarized our current understanding on the pathogen, the pathogenesis, and the rice resistance to the disease. Future work should pay attention to developing a more rapid and effective system to evaluate rice resistance and susceptibility to the disease, screening of rice germplasm for disease-resistance breeding, studying the resistance inheritance, and investigating the molecular mechanism of rice-false smut fungus interaction.

Establishment of Near Isogenic Lines of Huangzaosi Maize with Resistance against Maize Head Smut

[ Objective ] The paper was to establish the near isogenic lines of Huangzaosi maize with resistance against maize head smut, and to provide basis for its wide application in breeding and production. [Method] Combing with hybrid, backcress and self-cross method, as well as molecular markers, Huangzaosi maize was successfully introduced with head smut resistance, and its near isogenic lines were studied. The characteristics of resistant and susceptible variation and the changes of combining ability of near isogenic lines after backcrossing breeding were observed. [ Result ] The incidence rate of Huangzansi maize in control was 46% ; the incidence rates of the selected 24 near isogenic lines of resistant Huangzaosi were all lower than 10%, and the selected rate was 92.6% ; the combining ability of most near isogenic lines of resistant Huangzaosi maize had no significant difference with that of H^i maize in control; the yields of hybrids prepared by several inbred lines such as M135 and M140 were outstanding, which exceeded the control Zhengdan 958, showing a higher combining ability. [ Conclusion] The phenotype and combining ability of near isngenic lines of Huangzaosi maize with resistance against maize head smut obtained in the test were very close to Huan- gzaosi, and its resistance against maize head smut was greatly increased, thus solving the problems of Huangzaesi without resistance against maize head smut.

Analysis of Quantitative Trait Loci for Pathogenic Strain Pi-1 of Resistance to Rice False Smut in Rice(Oryza sativa)

In order to identify the resistant gene of rice false smut in rice, a recombi- nant inbred line （RILs） population with 157 lines derived from an inter-subspecies cross of Daguandao/IR28 by the single seed descent method was used to detect quantitative trait loci （QTLs） conferring resistance to strain Pi-1 of rice false smut caused by Usti/aginoiclea virens （Cooke） Takahashi in 2012 and 2013. The disease rate indexes of the two parents and 157 RILs caused by the strain Pi-1 of rice false smut were scored and the QTLs for rice false smut resistance were detected accordingly by QTL Cartographer software. Seven QTLs controlling false smut re- sistance were detected on chromosomes 2, 7, 8, 11 and 12, respectively, with the phenotypic variance of 8.5%-17.2%. There were four QTLs detected in 2012 and 2013, respectively, and only one QTL was found in both two years, the phenotypic variation explained by this individual QTL was 13.5% and 17.2%, and the additive effects of this QTL contributed to the 9.9% and 14.3% decrease of disease index and therefore the disease resistance increased. The direction of the additive effects at five loci qFsr2a, qFsr8a, qFsr8b, qFsr11 and qFsr12 coincided with that predicted by phenotypes of the parents, and the IR28 alleles at these loci had positive effect against rice false smut while the negative effects were found in Daguandao alleles at qFsr2b and qFsr7. The qFsr11 should be useful in rice breeding for resistance to rice false smut in marker-assisted selection （MAS） program.

Control of Maize Head Smut with 6% Tebuconazole FS

In order to test the control effect of 6% tebuconazole FS on maize head smut and to select its optimal dose for mixing seed, field tests were performed in 2012 and 2013. The results showed that 6% tebuconazole FS was effective to control maize head smut, and the control effect ranged from 73.4% to 91.1%. In addition, it also improved the maize yield by 36.7%-44.1%, compared with the blank control. The optimal dose of 6% tebuconazole FS for seed dressing was 166.5 g/100 kg to control head smut on maize.

Rice False Smut Fungus, <i>Ustilaginoidea virens</i>, Inhibits Pollen Germination and Degrades the Integuments of Rice Ovule

Ustilaginoidea virens is a flower-infecting fungus that forms false smut balls in rice panicle. Rice false smut has long been considered a minor disease, but recently it occurred frequently and emerged as a major disease in rice production. In vitro co-cultivation of U. virens strain with young rice panicles showed that U. virens enters inside of spikelets from the apex and then grows downward to infect floral organs. In response to U. virens infection, rice host exhibits elevated ROS accumulation and enhanced callose deposition. The secreted compounds of U. virens can suppress rice pollen germination. Examination of sectioning slides of freshly collected smut balls demonstrated that both pistil and stamens of rice flower are infected by U. virens, hyphae degraded the contents of the pollen cells, and also invaded the filaments. In addition, U. virens entered rice ovary through the thin-walled papillary cells of the stigma, then decomposed the integuments and infected the ovary. The invaded pathogen could not penetrate the epidermis and other layers of the ovary. Transverse section of the pedicel just below the smut balls showed that there were no fungal hyphae observed in the vascular bundles of the pedicel, implicating that U. virens is not a systemic flower-infecting fungus.

Inhibition of the spread of endophytic Sporisorium reilianum renders maize resistance to head smut

Head smut, caused by the fungal pathogen Sporisorium reilianum, poses a grave threat to maize(Zea mays) production worldwide. Here we report cytological and molecular evidence for maize resistance to head smut. During early stages of root infection, S. reilianum mycelium was capable of penetrating the root epidermis of both resistant(Ji1037) and susceptible(HZ4) inbred lines. S. reilianum hyphae were observed in the root–stem junction at 6 days after inoculation. In an attempt to monitor hyphal spread within the maize plant,a highly specific and sensitive real-time PCR method was established to estimate the hyphal content in infected maize tissues. During the upward growth of endophytic S.reilianum, the extent of hyphal spread was markedly different between Ji1037 and HZ4. Very little or no pathogen was detected in aerial parts of Ji1037, whereas large amounts of pathogen accumulated in aboveground tissues, particularly inflorescences, of HZ4. Thus,maize resistance to S. reilianum was achieved mainly by inhibition of endophytic hyphal growth rather than by prevention of early-root penetration by the pathogen.

Diversity in metagenomic sequences reveals new pathogenic fungus associated with smut in Job's tears

Smut is a serious disease in Job's tears,also known as adlay,and contributes to the reduction of crop yield in agricultural fields.In this study,the key pathogenic fungi in adlay smut disease were first identified by internal transcribed spacer region(ITS)high-throughput sequencing and then used to elucidate the composition and diversity of the fungal community in adlay smut.Results indicate that an abundance of operational taxonomic units(OTUs)were detected in the infected involucres of flowering plants and the OTUs were classified to nine phyla,20 classes,45 orders,90 families and 119 genera.A total of 4986 OTUs clustered together,sharing six core OTUs in all samples,while 145 OTUs were shared among four geographical groups.The Shannon and Simpson indices ranged from 0.137–1.629 and 0.357–0.970,respectively.Community diversity ranked as Anshun(AS)>Qinglong(QL)>Xingren(XR)>Xingyi(XY)among the four geographical groups by Shannon and Simpson indices,exhibiting complex community diversities among accessions and geographical groups.The richness and diversity data imply a weak relationship between the accession community richness and geographical origins of samples.Two closely related fungal genera,Sporisorium and Ustilago,were implicated as causes of smut disease.The genus Sporisorium appears to be more commonly found among accessions and thus is more likely to be the fungal pathogen causing smut in adlay.This work can facilitate strategies to control and prevent smut infection to improve adlay yield.

Study on Sugarcane Smut

The pathogen and characteristics, infection cycle, occurrence regularity and damage symptoms of sugarcane smut were introduced in the paper. Moreover, the comprehensive prevention measures were proposed on the basis of planting disease-resistant species, strengthening cultivation managements to improve dis- ease-resistant ability of sugarcane, and adopting fungicide control as assistant measures. The study provided certain basis for reducing damages of sugarcane smut and imorovin yield and quality of sugarcane.

Inheritance Pattern of Two-line Hybrid Rice Resistance to False Smut

[Objective] The aim of this study was to explore the inheritance pattern of the resistance of two-line hybrid rice to false smut. [Method] A moderate susceptible TGMS line 33S as the female parent, was crossed with 30 susceptible restorer lines and 23 resistant restorer lines. Then false smut resistance of the hybrid F1 and parents were identified. [Result] All F1 of 33S crossed with 30 susceptible re- storer lines were susceptible to rice false smut. The F1 of 33S crossed with 23 re- sistant restorer lines showed dominant, incompletely dominant or recessive inheri- tance. Dominant and incompletely dominant inheritance accounted for 87.0%. The diseased panicle rate, disease index, average diseased grains per panicle, and the highest diseased grains per panicle of F1 shared highly significant positive correla- tions with corresponding indicators of restorer lines. [Conclusion] This study provides references for genetic improvement of two-line hybrid rice.

Survey and examination of the potential alternative hosts of Villosiclava virens, the pathogen of rice false smut, in China

Rice false smut is caused by ascomycete Villosiclava virens, whose potential alternative hosts have been assumed previ- ously. Here its potential alternative hosts were surveyed and identified from 2008 to 2013 in the main rice-growing regions in China. Two common weeds in paddy fields, Digitaria sanguinalis Scop. and Echinochloa crusgalli （L.） Beauv., were found to present the similar symptoms to smut diseases in a few individuals in 2012 and 2013 in Zhejiang and Sichuan provinces of China, respectively. After the examinations of the spore morphology, their infection and extension mode in hosts, pathogen cell wall components, and molecular identification, the two pathogens were identified to be the Basidiomycetes, Ustilago syntherismae and Ustilago trichophora, respectively. So far there has been no alternative host of V. virens to be identified in China. These suggest that the alternative hosts of V. virens, if they do exist, are not possible to play an important role in the pathogen life cycle and the disease epidemics.

Research Progress and Prospect of Sugarcane Smut

Sugarcane smut disease( Ustilago scitaminea Sydow) is an important fungal disease worldwide,which seriously affects yield and quality of sugarcane and threatens the stability and development of sugarcane industry. This paper reviewed disease occurrence,damage,pathogen,symptoms,genetic diversity and detection methods of sugarcane smut,response of sugarcane to sugarcane smut stress and control measures of sugarcane smut. The in-depth research of sugarcane smut was also prospected,aiming to provide theoretical reference and scientific basis for the effective control of sugarcane smut.

Damage Characteristics and Control Measures of Maize Head Smut

Maize head smut is a worldwide disease, widely occurred in most maize fields. The disease had occurred in large area in the late 1970s, and became a limiting factor restricting the improvement of maize yield and total yield in China. After efforts of several generations of agricultural scientific workers, the symp- toms, pathogen, infection pathway, infection conditions, infection regularity and resistance genetic law of maize head smut have been studied. This paper introduces the incidence symptoms, pathogen sources, infection pathway, infection conditions, and physiological races of maize head smut, and put forwards the corresponding prevention measures, such as crop rotation, selection of resistant varieties, seed dressing, soil treatment with fungicides, promoting seedling emergence and strengthening field management.

Water Extract of Rice False Smut Balls Activates Nrf2/HO-1 and Apoptosis Pathways,Causing Liver Injury

Ustiloxins are vital cyclopeptide mycotoxins originally isolated from rice false smut balls that form in rice spikelets infected by the fungal pathogen Ustilaginoidea virens.The toxicity of the water extract of rice false smut balls(RBWE) remains to be investigated.Studies have shown that RBWE may be toxic to animals,but toxicological evidence is still lacking.In this study,we found that the IC50 values of RBWE to BNL CL.2 cells at 24 and 48 h were 40.02 and 30.11 μg/m L,respectively,with positive correlations with dose toxicity and time toxicity.After treatment with RBWE,the number of BNL CL.2 cells decreased significantly,and the morphology of BNL CL.2 cells showed atrophy and wall detachment.RBWE induced DNA presynthesis phase arrest of BNL CL.2 cells,increased the proportion of apoptotic cells and inhibited cell proliferation.RBWE up-regulated reactive oxygen species(ROS) levels and lowered mitochondrial membrane potentials.Additionally,Western blot and q RT-PCR results suggested that RBWE exerted the above effects by promoting the Nrf2/HO-1 and caspase-induced apoptosis pathways in vitro and in vivo.The contents of alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,and total bile acids in the serum of mice from Institute of Cancer were significantly up-regulated by RBWE.At the same time,RBWE can lead to increases in ROS and malondialdehyde contents,decreases in contents of oxidized glutathione,glutathione and reduced glutathione,as well as decrease in catalase and superoxide dismutase activities in mouse liver tissues,demonstrating that oxidative stress occurred in mice.Moreover,liver damage was further detected by haematoxylin-eosin staining and electron microscopy to verify the damage to the mice caused by RBWE.In general,RBWE may cause hepatotoxicity in vivo and in vitro via the apoptosis pathway,which provides a reference for hepatotoxicity and its mechanism of action.

Response of Sorghum Accessions against Newly Documented Pathotypes 5 and 6 of Head Smut Pathogen, <i>Sporisorium reilianum</i>

This study was conducted to identify resistance sources against the newly documented pathotypes (P5 and P6) of Sporisorium reilianum, causing sorghum head smut. A subset of 67 sorghum association panel (SAP) accessions, 29 in 2017 and 38 in 2018 along with checks BTx635 (resistant) and BTx643 (susceptible) were screened in the greenhouse against P5 and P6 pathotypes in two separate experiments in both years. At 18 to 20 days after planting, accessions were inoculated by injecting the seedlings below the apical meristem with sporidial suspensions following an established inoculation procedure. Three accessions (PI656091, PI533919, and PI533821) in 2017 and 17 accessions (PI597961, PI656071, PI656048, PI576435, PI534075, PI534145, PI656057, PI576437, PI651492, PI656014, PI656025, PI655999, PI656010, PI656082, PI534123, PI533927, PI656100) in 2018 were identified as potential resistant sources to both P5 and P6 pathotypes of S. reilianum. These SAP accessions are new sources that can be further used to develop new breeding parental lines and hybrids and mapping populations for quantitative trait loci markers closely linked to head smut resistance.

A PCR-Based Method for Detecting Pathogen of Rice False Smut

Rice false smut is a destructive disease that affects rice grain badly.The disease seriously affects the yield and quality of rice in Heilongjiang Province.In this paper,a pair of specific primers was designed to detect the false smut pathogen rapidly and efficiently.The results showed that the pair of primers had strong specificity for false smut pathogen.In addition,the sensitivity of this primer to the genomic DNA of rice false smut pathogen in PCR reaction was 1 pg.By using these primers,the rice false smut pathogen could be detected within 48 h after inoculation,and a PCR reaction system with good specificity and high sensitivity was established.

Investigation on Outbreak of Sesamia inferens and Smut in Cane-growing Regions of Yunnan Province

[Objectives]This study was conducted to clarify the outbreak and disaster characteristics of Sesamia inferens Walker and smut in cane-growing regions in the low-latitude plateau of Yunnan Province,where there is continuous high temperature and drought in spring and summer.[Methods]A systematic investigation and analysis was conducted on different varieties in different types of fields in 16 cane-growing regions with outbreak of S.inferens and smut in Yunnan from 2019 to 2021.[Results]The outbreak of S.inferens and smut in cane-growing regions in the low-latitude plateau of Yunnan Province resulted in an average percent of dry hearts in the range of 17.3%-56.3%,and as high as 84.0%in severe cases.70%thiamethoxam seed treatment dispersible powder and clothianidin or thiamethoxam fertilizer granules were ineffective against S.inferens,while 10%monosultap·thiamethoxam or 10%clothianidin·monosultap granules had a significant control efficacy on S.inferens.The average diseased plant rate ranged from 14.8%to 89.7%in susceptible varieties with smut,and was as high as 100%in severe cases.ROC 1,Yuetang 93-159,Yuetang 00-236,and Liucheng 05-136 had strong resistance,with an average diseased plant rate of 0-5.5%and the highest rate of 7.6%.S.inferens and smut both broke out in cane-growing regions with an altitude below 1200 m.It is recommended to pay attention to disease and pest monitoring in cane-growing regions with an altitude below 1200 m,and combine it with the management of new plants and ratoon plants to effectively prevent and control the first and second generation of S.inferens-infested seedlings from March to May.It is also recommended to strengthen the promotion and application of excellent disease-resistant varieties,so as to achieve a reasonable distribution of varieties and fundamentally control the outbreak and epidemic of sugarcane smut.[Conclusions]This study provides a basis for the effective prevention and control of catastrophic S.inferens and smut caused by extreme climate.

A maize WAK-SnRK1a2-WRKY module regulates nutrient availability to defend against head smut disease

Obligate biotrophs depend on living hosts for nutrient acquisition to complete their life cycle,yet the mechanisms by which hosts restrict nutrient availability to pathogens remain largely unknown.The fungal pathogen Sporisorium reilianum infects maize seedlings and causes head smut disease in inflorescences at maturity,while a cell wall-associated kinase,ZmWAK,provides quantitative resistance against it.In this study,we demonstrate that S.reilianum can rapidly activate ZmWAK kinase activity,which is sustained by the 407th threonine residue in the juxtamembrane domain,enabling it to interact with and phosphorylate ZmSnRK1a2,a conserved sucrose non-fermenting-related kinase a subunit.The activated ZmSnRK1a2 translocates from the cytoplasm to the nucleus,where it phosphorylates and destabilizes the transcription factor ZmWRKY53.The reduced ZmWRKY53 abundance leads to the downregulation of genes involved in transmembrane transport and carbohydrate metabolism,resulting in nutrient starvation for S.reilianum in the apoplast.Collectively,our study uncovers a WAK-SnRK1a2-WRKY53 signaling module in maize that conveys phosphorylation cascades from the plasma membrane to the nucleus to confer plant resistance against head smut in maize,offering new insights and potential targets for crop disease management.

Overproduction of OsRACK1A,an effector-targeted scaffold protein promoting OsRBOHB-mediated ROS production,confers rice floral resistance to false smut disease without yield penalty

Grain formation is fundamental for crop yield but is vulnerable to abiotic and biotic stresses.Rice grain production is threatened by the false smut fungus Ustilaginoidea virens,which specifically infects rice floral organs,disrupting fertilization and seed formation.However,little is known about the molecular mechanisms of the U.virens-rice interaction and the genetic basis of floral resistance.Here,we report that U.virens secretes a cytoplasmic effector,UvCBP1,to facilitate infection of rice flowers.Mechanistically,UvCBP1 interacts with the rice scaffold protein OsRACK1A and competes its interaction with the reduced nicotinamide adenine dinucleotide phosphate oxidase OsRBOHB,leading to inhibition of reactive oxygen species(ROS)production.Although the analysis of natural variation revealed no OsRACK1A variants that could avoid being targeted by UvCBP1,expression levels of OsRACK1A are correlated with field resistance against U.virens in rice germplasm.Overproduction of OsRACK1A restores the OsRACK1A-OsRBOHB association and promotes OsRBOHB phosphorylation to enhance ROS production,conferring rice floral resistance to U.virens without yield penalty.Taken together,our findings reveal a new pathogenic mechanism mediated by an essential effector from a flower-specific pathogen and provide a valuable genetic resource for balancing disease resistance and crop yield.