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.

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.

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.

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.

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.

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.

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.

CONTROL TECHNIQUE OF BACTERIAL CANKER OF POPLAR

The poplar bacterial canker caused by Erwinia sp. is a serious disease spreading in most parts of Northeast China, where poplars are cultivated. The disease seriously infects those poplars belonging to section Aigeiros or the intersection hybrids of section Tacamahaca and section Aigeiros and their clones. Five fine poplar cultivars resistant to the bacterial canker have been obtained by screening tests. They are Lz2(Populus simonii × nigra var. italica), A15(P. × sp.), 613(P. × sp.), A102and A98(P. × simonii × sp.). The following chemicals are obviously effective, such as: 50% DT germicide, 40% XF-136 and 10% C.C.M.A. or 12.5% ramphencol and 50,000 ppm/ml validamycin, with which the stems or the roots can be watered.

Control Efficacy of Streptomyces sp. SY-L12 and Chitooligosaccharide on Kiwifruit Bacterial Canker, Pseudomonas syringae pv. actinidiae

[ Objective] The paper was to develop a priority scheme for prevention and control of kiwifruit bacterial canker with biological agents. [ Method ] The inhibitory effect of Streptomyces sp. SY-L12 on the growth of Pseudomonas syringae pv. actinidiae were assessed using liquid co-culture and inhibition zone meth- ods. The control efficiency of Streptomyces sp. fermentation broth against kiwifruit bacterial canker was studied via potted trial. [ Result ] As Streptomyces sp. SY- L12 was incubated for 24 h, the growth inhibition rate of its fermentation broth on P. syr/ngae pv. actinidiae was 94.0%. The control efficiency of fermentation broth against P. syringae pv. actinidiae was the best of 85.4% in potted trial when Streptomyces sp. SY-L12 grew at 28~C, 150 rpm for 8 -9 d, better than that of control agent copper hydroxide. The control efficiency was improved as the agent was mixed with 1 mg/L chitooligosaccharide. [ Conclusion] Streptomyces sp. SY-L12 could effectively inhibit the growth of P. syr/ngae pv. actinidiae, and can be developed as biological agent against kiwifruit bacterial canker.

Indoor Screening of Bactericides against Kiwifruit Bacterial Canker

[ Objective ] The paper was to screen the effective agents against kiwifruit bacterial canker in Wanzhou District of China. [ Method ] Ten kinds of com- mon commercial bactericides preliminarily screened were selected for indoor antibacterial test using paper plate method and plate streaking method, and six kinds of bactericides with good antibacterial effects were further screened for indoor toxicity test. [ Result] Chloramphenicoloethylicin had the best antibacterial effect, with EC50 of 36.34 rag/L; followed by Xintianxlucai No. 6 and embamycin, with EC50 of 83.21 and 92.34 mg/L, respectively. [ Conclusion] Cldoramphenicoloethylicin, Xintianxiucai No. 6 and embamycin screened in the test had good antibacterial effect against kiwifruit bacteriai canker in Wanzhou District, and chlorampheni- coleethylicin was the most effective agent. These three agents could be directly applied to control kiwifruit bacterial canker in production, while application time and climate conditions in fields should also be considered during the actual application process. It is suggested that three agents can be used alternatively in order to de- lay the resistance of plants.

Unveiling an important interaction in forestry:Ectomyelois muriscis and Khaya grandifoliola cankers and tree growth

African mahogany,Khaya grandifoliola C.DC.,is the most planted mahogany species in Brazil,and became the choice species when plantations of Brazilian mahogany,Swietenia macrophyla King became unsustainable due to their susceptibility to the shoot borer Hypsipyla grandella Zeller.This study evaluated plantation growth and the occurrence of cankers associated with Ectomyelois muriscis Dyar larvae.Surveys recorded plantation volume,survival,and canker incidence of a plantation established in 2010 at 6 m×5.5 m spacing.Larvae were collected and reared on canker bark fragments until final adult stage.Cankers were frequently associated with Ectomyelois muriscis larvae.The average incidence of the moth(2.9%)was not a significant concern,however,an extreme incidence of 45%would indicate that management strategies should be developed.This is the first report of Ectomyelois muriscis attacking the bark of Khaya grandifoliola in the Cerrado biome.

Occurrence Regularity and Integrated Control Technology of Canker in Citrus

Canker is a quarantine bacterial disease that seriously harms leaves,branches and fruits of citrus,leading to a decrease in the production and affecting the commodity and sales of citrus.Citrus canker has the characteristics of fast spreading speed and difficult radical cure.Through the identification of symptoms and a summary of occurrence regularity,the integrated prevention and control technology for citrus canker is described in this article,in order to achieve effective prevention and control and reduce prevention and control cost.

Damage and Control of Blueberry Canker

As the most common and serious disease of blueberry,blueberry canker has caused huge economic losses to the blueberry planting industry.This paper summarized the pathogenic bacteria,symptoms,regularities and prevention methods of blueberry canker.

Characterization of Chinese Isolates of Mango Bacterial Canker Xanthomonas campestris pv. mangiferaeindicae

Mango bacterial canker is caused by Xanthomonas campestris pv. mangiferaeindicae. During 2009 and 2013,leaves,twigs and fruits of mango were collected from commercial and experimental mango fields with typical canker symptoms in Hainan,Guangxi,Guangdong and Szechwan Provinces of China. The causal agent was identified as X. campestris pv. mangiferaeindicae through KC semi-selective medium isolation,pathogenicity tests,and sequencing of the gyrB gene.

Preliminary Study on the Control of Kiwifruit Canker by 6%Ascorbic Acid Water Aqua

[Objectives]This study was conducted to investigate the control efficacy of 6% ascorbic acid water aqua on kiwifruit bacterial canker. [Methods]The ascorbic acid water aqua was sprayed onto annual potted kiwifruit seedlings once every 5 d,for three times in total,and Pseudomonas syringae pv. Actinidiae was inoculated by needle punching 7 d later. Scab diameter and number of newly infected scab were investigated to analyze the control efficacy. [Results] Kiwifruit plants were treated with 6% ascorbic acid water aqua,and after 30 d,the 20 mg/L treatment showed an incidence rate of 25. 5%,and the 60 mg/L treatment exhibited an incidence rate only of 12. 1%; and after 60 d,the relative control efficacy reached 90. 7% at the mass concentration of 60 mg/L. [Conclusions] The6% ascorbic acid water aqua weakened the destruction and expansion of the pathogen in plants,effectively improved the resistance in kiwifruit plants,and had certain control efficacy on kiwifruit canker.