High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Evaluation of Resistance of Different Kiwifruit Varieties (Lines) to Canker Disease and Brown Spot Disease

Kiwifruit canker and brown spot are significant diseases affecting kiwis,caused by Pseudomonas syringae patho-genic variations(Pseudomonas syringae pv.Actinidiae(Psa))and Corynesporapolytica(Corynespora cassiicola).At present,the research on canker disease and brown spot disease mainly focuses on the isolation and identification of pathogenic bacteria,drug control,resistance gene mining and functional verification.Practice has proved that breeding disease resistant varieties are an effective method to control canker disease and brown spot disease.However,most existing cultivars lack genes for canker and brown spot resistance.Wild kiwifruit resources in nat-ure exhibit extensive genetic diversity due to prolonged natural selection,containing numerous resistance genes.But,due to insufficient understanding of the resistance of most kiwifruit varieties(lines)to canker disease and brown spot disease,some high-quality resources have not been fully utilized.The incidence of canker and brown spot of 18 kiwifruit cultivars(lines)was measured by inoculating isolated branches and leaves,and their resistance to canker and brown spot was analyzed according to the length,disease index,mean diameter,and systematic clustering.The results were as follows:Among 18 different kiwifruit varieties(lines)for canker disease,there were two highly resistant materials,eight disease-resistant materials,four disease-susceptible materials,and two highly susceptible materials.Moreover,regarding brown spot disease,there were one highly resistant material,five dis-ease-resistant materials,four susceptible materials,and three highly susceptible materials.Furthermore,four resources were resistant to both diseases.The outcomes provided a theoretical basis for breeding kiwifruit against canker and brown spot.

Systematic analysis and functional verification of citrus glutathione S-transferases reveals that CsGSTF1 and CsGSTU18contribute negatively to citrus bacterial canker

Citrus bacterial canker(CBC) is resulted from Xanthomonas citri subsp. citri(Xcc) infection and poses a significant threat to citrus production.Glutathione S-transferases(GSTs) are critical in maintaining redox homeostasis in plants, especially in relation to abiotic and biotic stress responses. However, the function of GSTs in resisting CBC remains unclear. Here, citrus glutathione S-transferases were investigated applying a genome-wide approach. In total, 69 CsGSTs belonging to seven classes were identified, and the phylogeny, chromosomal distribution, gene structures and conserved motifs were analyzed. Several CsGSTs responded to Xcc infection, as observed in the upregulation of CsGSTF1 and CsGSTU18 in the CBC-sensitive ‘Wanjincheng' variety but not in the resistant ‘Kumquat' variety. CsGSTF1 and CsGSTU18 were localized at the cytoplasm. Transient overexpression of CsGSTF1 and CsGSTU18 mediated reactive oxygen species(ROS) scavenging, whereas the virus-induced gene silencing(VIGS) of CsGSTF1 and CsGSTU18 caused strong CBC resistance and ROS burst. The present study investigated the characterization of citrus GST gene family, and discovered that CsGSTF1 and CsGSTU18 negatively contributed to CBC through modulating ROS homeostasis. These findings emphasize the significance of GSTs in infection resistance in plants.

An endolysin gene from Candidatus Liberibacter asiaticus confers dual resistance to huanglongbing and citrus canker

The most damaging citrus diseases are Huanglongbing(HLB)and citrus canker,which are caused by Candidatus Liberibacter asiaticus(CaLas)and Xanthomonas citri pv.citri(Xcc),respectively.Endolysins from bacteriophages are a possible option for disease resistance in plant breeding.Here,we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas.LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc,according to inhibition zone analyses.The two genes,driven by a strong promoter from Cauliflower mosaic virus,35S,were integrated into Carrizo citrange via Agrobacterium-mediated transformation.More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB,allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term.LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc,indicating that LasLYS2 had dual resistance to HLB and citrus canker.A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations,which might boost the citrus defense response,according to transcriptome analysis.We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2.Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2.Overall,the work shed light on the mechanisms of resistance of CaLas-derived endolysins,providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.

Investigation of Kiwifruit Bacterial Canker Disease(Pseudomonas syringae pv.Actinidiae Takikawa) in Guanzhong Area of Shanxi Province and Its Biological Control

[ Objective ] The paper was to grasp the incidence characteristic of kiwifruit bacterial canker disease （Pseudomonas syrirtgate pv. actinidiae Takikawa） in Guanzhong area of Shanxi province, and to screen the endophytic actinomycetes with control effect against kiwifruit bacterial canker. [ Method] The incidence characteristics of kiwifruit bacterial canker disease in 65 orchards of 13 villages in guanzhong area of Shanxi province （Yangling, Zhouzhi, Meixian） were investiga- ted and analyzed. The antagonistic strains against Pseudomonas syringae were simultaneously screened from 242 endophytic actinomycetes, and carried out field dis- ease control test. [ Result] The average incidence rate of kiwifruit bacterial canker disease in guan^ong area of Shanxi province was 7.95% ; the variety of kiwifruit had dose relationship with incidence rate. 76 strains bad inhibition activity agmnst P. syringae, while the inhibition zone diameters of fermentation broths of 35 strains against P. syr/ngae were greater than 20 nun. TIASA5 strain had inhibition activity against four target bacteria; gCLA4 strain not only had inhibition activity against four target bacteria, but also had strong inhibition activity against 10 species of plant pathogenic fungi. Field test showed that the fermentation broth of gCLA4 strain had obvious control effect against kiwifruit bacterial canker disease, and the relative control effect at 21 d could reach 66.7%. [ Conclusion] The an- tibacterial spectrum of fermentation broth of gCLA4 strain is broad, so gCLA4 strain has the potential to be developed into a broad-spectrum agricultural fungicide. Key words Kiwifruit bacterial canker disease; Endophytic actinomycetes; Biological control; Field test

Outbreak and the Reasons of Apple Valsa Canker in Yantai Apple Producing Area in 2011

[Objective] This study aimed to investigate an outbreak of apple valsa canker （Valsa ceratosperma） in Yantai apple producing area in 2011 and analyze the major causes of the disease. [Method] In May 2011, 21 commercial apple or-chards in Qixia, Haiyang and Laiyang were selected to investigate the outbreak of applevalsa canker. [Result]The results showed that apple canker occurred seriously in Yantai apple producing area in 2011. The ratio of diseased plants with new canker scars was 68.20% and the ratio of dead plants infected with Valsa cer-atosperma was 2.76%. The average ratios of diseased branches and one-year-old dead branches were 23.98% and 10.74%, respectively. The percentage of orchards with more than 50% diseased plants accounted for 25%-30% of the total number of orchards investigated, and the overal prevalence situation was more serious than normal years. In the investigation, 967 new canker scars were observed, with an average of 2.32 canker scars per plant. Specifical y, 80.04% canker scars were de-veloped from pruning wounds; 60.29% canker scars were developed from previous scars. [Conclusion] The long-period precipitation in the autumn of 2010, low temper-ature in the winter of 2010 and the severe drought in the spring of 2011 might be the major factors causing the outbreak of apple valsa canker in Yantai apple pro-ducing area in 2011. Pruning wounds were the main infection entrances of V. cer-atosperma, and the recurrence in previous scars was the main reason for the out-break of apple canker in spring.

Regional epidemic laws of poplar Ice Nucleation Active bacterial canker

Through the methods of correlation analysis and main factor analysis, the relationship between the poplar INA bacte-rial canker and circumstances was analyzed and 9 main factors for affecting the disease were selected. Based on the compre-hensive analysis of main factors and induced factors, the standard for risk grades of this disease was promoted and northeast region of China was divided into 4 districts with different risk grades: seriously occurring district, commonly occurring district, occasionally occurring district, and un-occurring district. Nonlinear regression analysis for six model curves showed that the Richard growth model was suitable for describing the temporal dynamics of poplar INA bacterial canker. By stepwise variable selection method, the multi-variable linear regression forecasting equation was set up to predict the next year抯 disease index, and the GM (1,1) model was also set up by grey method to submit middle or long period forecast.

Inhibitory Effect of Antifungal Substances from Chaetomium globosum ND35 on Pathogenic Fungi of Valsa canker

[Objective] Inhibition mechanism of the antifungal substances from Chaetomium globosum ND35 against pathogenic fungi of Valsa canker was investigated.[Method] The inhibitory effect of antifungal substances （AFS） produced by endophytic C. globosum ND35 on pathogenic fungi of Valsa canker was studied by means of dual culture,extraction,thin layer chromatography and bioassay of antifungal activity. [Result] The crude extracts of AFS could strongly inhibit Valsa sordida and V. mali. The suppression percentage of mycelial growth of two pathogenic fungi were 66.4% and 72.6%,respectively. The inhibition percentages of conidia germination of two pathogenic fungi were 92.2% and 80.4%,respectively. Separation of thin layer chromatography and bioassay of antifungal activity indicated that the fraction No.2 of AFS played an important role in the process of inhibition on pathogenic fungi,causal agents of Valsa canker. [Conclusion] The antifungal substances from C. globosum ND35 is potential for biological control on Valsa canker.

Bio-control trials of Chaetomium spirale ND35 against apple canker

A new endophytic antagonistic fungus, Chaetomium spirale ND35 from Populus tomentosa, was reported. The bio-control trials of C. spirale ND35 against the Valsa Canker of apple were preliminarily investigated. The results of dual culture on PDA plate showed that C. spirale ND35 was capable of strong antagonism against Valsa ceratosperma, and for inhibiting the mycelial growth of V. ceratosperma,.the crude extract of liquid culture of corn steep powder broth was more effective than that one of malt extract broth (MEB). The results of bio-control in greenhouse and field indicated that the disease incidence of apple tree treated with C. spirale ND35 was lower significantly than that treated by other methods. The re-isolation experiment suggested that C. spirale ND35 could colonize in stems and branches of apple trees successfully, and the ND35 colonization rate of the treatment with solid wheat bran culture was higher than that of corn steep powder broth, but the field experiment result the control effect of liquid culture of C. spirale ND35 was better than that of solid culture.

猕猴桃细菌溃疡病(Actinidia bacterial canker)病原细菌鉴定

对四川省苍溪县猕猴桃的细菌病害进行系统鉴定表明,该病属细菌溃疡病,其病原细菌为 Pseudomonas syringae Pv.actinidea。病原细菌的鉴定属国内首次报导.

The influences of biotic and abiotic factors on the occurrence and severity of poplar canker disease in Qingfeng County, China and the management implications

Landscape pathology is a research approach that can provide validation of the effectiveness of regional controls of forest disease at a landscape scale. In this paper, we analyzed the effects of stand features, management approaches, and geographical locations on poplar canker disease incidence （DI） and disease severity index （DSI） of individual trees at a 10 km x 10 km mesoscale landscape in Qingfeng County, China. DI varied significantly with stand age, tree densities, and the degree of canopy closure. DI in stands younger than 4 years old was significantly lower than that in the stands over 6 years old and reached the highest value at a stand age of 8-10 years. Overall, DI was positively correlated with stand age, stand density, andthe degree of canopy closure. DI was significantly lower in agro-forest stand patches than in other three patch types, i.e. isolated patch, pure stand patch, and mixed stand patch. Poplar plantations distributed around and near to villages exhibited significantly higher DI mainly due to human activities and herbivores. Fragmentation or connectivity in this mesoscale landscape seemed not impact disease occurrence. DSI was not significantly correlated with stand density, but varied significantly with tree varieties and trees ages. DSI was highest in stands of 10-12 year trees for all poplar varieties we studied here. Plantation density and plantation age were thus critical factors in determining DI and DSI. A logistic predictive model of disease occurrence was developed for the study area, considering varieties, age, height, density, canopy cover, stand types, patch types, management status, and stand geographical locations. Our study here shows that adjustment of stand density by thinning at different plantation ages is an effective approach controlling the occurrence canker disease in short-rotation poplar plantations at the landscape scale.

Bleeding canker of pears caused by Dickeya fangzhongdai:Symptoms,etiology and biology

Bleeding canker,a devastating disease of pear trees(Pyrus pyrifolia L.),was first reported in the 1970 s in Jiangsu,China and more recently in other provinces in China.Trees infected with bleeding canker pathogen,Dickeya fangzhongdai,develop cankers on the trunks and branches,and a rust-colored mixture of bacterial ooze and tree sap could be seen all over the trunks and branches.In this study,we provided detail descriptions of the symptoms and epidemiology of bleeding canker disease.Based on pathogenic and phenotypic characterizations,we identified the causal agent of bleeding canker of pear as D.fangzhongdai.Dickeya fangzhongdai strains isolated from pear were also pathogenic on Solanum tuberosum,Brassica pekinensis,Lycopersicon esculentum,and Phalaenopsis aphrodite based on artificial inoculation,and the pathogen were more virulent on potato than that of D.solani strain.This study provides new information about this disease and bleeding canker disease of pear.

Transcription factor WRKY22 regulates canker susceptibility in sweet orange (Citrus sinensis Osbeck) by enhancing cell enlargement and CsLOB1 expression

Pathological hypertrophy(cell enlargement)plays an important role in the development of citrus canker,but its regulators are largely unknown.Although WRKY22 is known to be involved in pathogen-triggered immunity and positively regulates resistance to bacterial pathogens in Arabidopsis,rice and pepper,the CRISPR/Cas9-mediated partial knockout of CsWRKY22 improves resistance to Xanthomonas citri subsp.citri(Xcc)in Wanjincheng orange(Citrus sinensis Osbeck).Here,we demonstrate that CsWRKY22 is a nucleus-localized transcriptional activator.CsWRKY22-overexpressing plants exhibited dwarf phenotypes that had wrinkled and thickened leaves and were more sensitive to Xcc,whereas CsWRKY22-silenced plants showed no visible phenotype changes and were more resistant to Xcc.Microscopic observations revealed that the overexpression of CsWRKY22 increased cell size in the spongy mesophyll.Transcriptome analysis showed that cell growth-related pathways,such as the auxin and brassinosteroid hormonal signaling and cell wall organization and biogenesis pathways,were significantly upregulated upon CsWRKY22 overexpression.Interestingly,CsWRKY22 activated the expression of CsLOB1,which is a key gene regulating susceptibility to citrus canker.We further confirmed that CsWRKY22 bound directly to the W-boxes just upstream of the transcription start site of CsLOB1 in vivo and in vitro.We conclude that CsWRKY22 enhances susceptibility to citrus canker by promoting host hypertrophy and CsLOB1 expression.Thus,our study provides new insights into the mechanism regulating pathological hypertrophy and the function of WRKY22 in citrus.

Production of Transgenic Anliucheng Sweet Orange(Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR （pattern recognition receptor） gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene （HPT） was introduced into Anliucheng sweet orange （Citrus sinensis Osbeck） via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin （HYG） and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identiifed by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was brielfy discussed.

Real-time PCR assay for detection of Dickeya fangzhongdai causing bleeding canker of pear disease in China

Bleeding canker, caused by Dickeya fangzhongdai, is a devastating disease of pear in China. The bacterium causes cankers, branch die-back, and eventually kills pear trees. The typical sign of bleeding canker infection is a rusty-brown bacterial ooze that exudes down from cankers onto branches or trunks. However, early symptoms and signs are inconspicuous, which makes effective disease management difficult. Detection and identification of D. fangzhongdai are time-consuming and difficult because no rapid method exists to date. In this study, a Taq Man real-time PCR assay was developed for D. fangzhongdai based on an elongation factor G(fus A) gene. The real-time PCR assay detected 0.2 pg μL^-1 DNA and 1×10^3 Cfu m L^-1 of D. fangzhongdai. Based on this assay, bleeding canker on asymptomatic pear trees can be diagnosed as early as 5 days after infection. The real-time PCR assay can facilitate disease management by providing early and accurate diagnosis of the bleeding canker disease of pear.

Identification of Citrus Canker on Citrus Leaves and Fruit Surfaces in the Grove Using Deep Learning Neural Networks

Citrus canker(Xanthomonas axonopodis pv.citri)is a bacterial disease of citrus tree leaves and is the most feared citrus disease,affecting all types of important citrus crops.Currently,there is no cure for citrus canker but experimental prevention methods.Citrus canker is a quarantine disease,meaning that citrus fruits with citrus canker cannot be transported between countries or markets,and this is a huge threat to the citrus production industry.Citrus exports to international markets are highly restricted today due to fruit diseases such as citrus canker because fruits are quarantined for bigger world markets such as European Union(EU)and United States of America citrus markets.For a row-guided robot to detect citrus canker infections on leaves and fruits in the grove,there is a need for an accurate method that can detect defects on the leaves and surface of fruits,classify citrus leaf and fruit images on-the-go,and discriminate citrus canker from other defects.Previous methods in machine vision could detect defects on the surface of citrus fruits,though,the methods were unable to differentiate defects.The neural network system invents edge detection,and automatically learns to characterize the image in terms of edges that appear in the image,and give a more succinct,higher-level representation than raw pixels.A standard strategy in deep learning neural networks is to run the learning algorithm with many optimization parameters and pick the model that gives the best performance on a validation set.The authors are optimistic that identification of citrus canker on citrus leaves and fruit surfaces in the grove could be one of the agricultural problems that can potentially benefit from deep learning neural network approach,and consequently,help to eradicate the predicaments facing the citrus industry.

Factors Influencing Bacterial Canker Disease of Kiwifruit and the Control Countermeasures

Survey was conducted to investigate the occurrence laws of bacterial canker disease of kiwifruits in Liupanshui area, Guizhou Province, and the patho- gens and hazard symptoms of the canker disease were introduced. The results showed that the factors influencing the occurrence of bacterial canker disease of ki- wifruit were identified as inter-species resistance, agronomic traits, meteorological and ecological conditions, cultivation and management techniques and fertilization levels. Techniques for the comprehensive control of kiwi canker disease were stated, and control suggestions were proposed.

Control Effects of 9 Fungicides on Citrus Canker

In order to screen out effective fungicides for controlling citrus canker,the control effects of 9 fungicides on Jinhong bingtang orange canker were studied.The results showed that 77%cupric calcium sulfate WP,30%copper oxychloride SC,46%copper hydroxide WG,30%thiodiazole-copper SC and 40%zinc thiazole SC had better comprehensive control effect on citrus canker,6%benziothiazolinone WG had an average effect,3%zhongshengmycin AS,2%kasugamycin AS and 33.5%oxine-copper SC had poor control effect.Therefore,fungicides could be used alternately or choose compounded preparation.In addition,adjuvants could be considered in use,which was an effective way to enhance efficacy,reduced dosage and delayed pesticide resistance.

STUDY ON THE HOST BIOLOGICAL INDUCED RESISTANCE OF BACTERIAL CANKER OF POPLAR

This is the first time that biological induced resistance has been used in the research of the bacterial canker on poplar. After inoculated with less-aggressive strains, middle-aggressive strains and non-aggressive strains (high-aggressive strains have been treated with 400W ultraviolet lamp for 30 min on super-clean working table) poplar seedlings have been inoculated with high-aggressive strams. the results of those experiments shown that strain 3 (from Suihua, Heilongjiang)and strain 9 (from Liaoning) can significantly enhance the activities of the peroxidase of host. and the efects of twice inoculation were much better than that of once inoculation. 7. 1 % and 4.0% of the peroxidase activities have been enhanced respectively in the seedlings which were twice inoculation with strains 3 and strains 9. 1. 1 % and 1 .2 % of the peroxidase activities have been enhanced by inoculated strains 3 and strains 9 once into seedlings.

BACTERIAL SWOLLEN STEM CANKER ON POPLAR:THE DISEASE AND ITS CAUSES

The bacterial swollen stem canker of poplar is prevalent in planted poplars of northeast China. Serious damage occurs in the form of necrosis, putridity, cankers, swollen stems and discolored wood of trunks and branches of young and mature trees. Tests of allergic reaction, pathogenicity, culture characteristic, Gram stain and anaerobic culture revealed that the pathogen is Erwinia sp. Its occurrence depends mainly on the predisposing factors of the disease among which the susceptibility of poplars is the most important one. The disease becomes widespread in association with low-lying and high-humidity terrain, frost injury, node wounds, insect damage, and pruning and other mechanical wounds. Harmful factors promote the disease.