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 thei...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.展开更多
Fungal pathogens causing poplar canker diseases are cosmopolitan in species,and have a wide range of hosts.These pathogens have diverse anamorphs and their morphology overlaps with each other;Their teleomorphs are har...Fungal pathogens causing poplar canker diseases are cosmopolitan in species,and have a wide range of hosts.These pathogens have diverse anamorphs and their morphology overlaps with each other;Their teleomorphs are hardly discovered in nature.Furthermore,the identification of these pathogens is usually limited by the geography,host and taxonomic knowledge.Therefore,a culture-independent method used to identify determine pathogens is expected to be developed for field diagnostics.Through amplifying,sequencing and analyzing multiplex nucleic acid templates and genetic marked target sequences,a multiplex PCR technique has been established and used to directly detect various environmental samples.In this study, four pathogen strains and environmental samples were amplified using fungal universal primers ITS1/ ITS4 and EF1-728F/EF1-986R by general PCR and multiplex PCR.The amplicons were sequenced,and then aligned in GenBank.The result showed that the multiplex PCR was able to successfully amplify the target gene and identify the pathogens from environmental samples in the condition of an annealing temperature of 55.6℃and primers final concentration of 0.2μmol·L<sup>-1</sup>.The multiplex PCR could amplify the target at the concentration level of approximately lng of genomic DNA.This method would provide a useful tool for the identification of canker pathogens by the multiplex PCR and the high-throughput DNA microarray detection of environmental samples.展开更多
Ambient temperature affects the occurrence and prevalence of plant disease.Most bacterial diseases are damaging at high temperatures.However,kiwifruit bacterial canker caused by Pseudomonas syringae pv.actinidiae(Psa)...Ambient temperature affects the occurrence and prevalence of plant disease.Most bacterial diseases are damaging at high temperatures.However,kiwifruit bacterial canker caused by Pseudomonas syringae pv.actinidiae(Psa)has been found to be prevalent at relatively cool temperatures,and it is unclear how ambient temperature affects the development of kiwifruit bacterial canker.In this study,basal resistance to Psa was suppressed in kiwifruit at cool growth temperature(16℃)compared with at normal temperature(24℃).In addition,RNA sequence analysis and ethylene content assessment indicated that ethylene modulated kiwifruit resistance to Psa at normal growth temperature and that cool temperature inhibited ethylene accumulation and Psa-induced activation of the ethylene signaling pathway in kiwifruit.Virusmediated silencing of the kiwifruit ethylene signaling gene AcEIN2 suppressed kiwifruit resistance to Psa at normal growth temperature.Exogenous application of ethylene inhibitor 1-methylcyclopropene eliminated the difference in kiwifruit resistance to Psa at 16 and 24℃.Exogenous application of ethylene analogues ethephon induced resistance to Psa in kiwifruit.In conclusion,cool temperatures impair basal resistance to Psa by reducing the activation of ethylene biosynthesis and signaling in kiwifruit.The results provide clues for new strategies to control plant diseases in a context of global environmental change.展开更多
The p H plays a key role in the growth and colonization of plant pathogens as well as the onset and progression of the symptoms they cause within the host.Plants may quickly alter their apoplastic p H(pH_(apo))to prot...The p H plays a key role in the growth and colonization of plant pathogens as well as the onset and progression of the symptoms they cause within the host.Plants may quickly alter their apoplastic p H(pH_(apo))to protect themselves against infection.However,pathogens can also alter the p H of their ambient environment to promote their own growth.Citrus canker is a serious plant disease caused by Xanthomonas citri subsp.citri(Xcc).This Gram-negative aerobic rod is usually cultured in Luria-Bertani(LB)medium at p H 7.However,little is known about the changes in p H both in this medium as Xcc grows and in the leaf apoplast in response to Xcc infection and colonization.Moreover,the differences in leaf apoplast p H between Xcc-resistant and Xcc-susceptible citrus genotypes are also unknown.Here,Xcc grew well in liquid LB medium at initial p H6-8 and the pathogen altered the medium p H to 6.8±0.4.Xcc growth declined at p H 5 and was zero at p H 3,4,9,and 10.In susceptible sweet orange infected with Xcc inoculum,canker symptoms were inhibited at p H 3,4,and 10 but did not differ in the range of p H 5-9.As expected,canker symptoms were absent at all inoculum p H in the resistant Citron C-05.For both genotypes,Xcc only grew well in the leaves exposed to p H 5-8 inoculums.At four days post-inoculation(4 dpi),the foliar pH_(apo)of resistant Citron C-05 had rapidly declined from 5.6 to 4.4.At 2 dpi,the p Hapoof susceptible sweet orange had rapidly increased from 5.6 to 6.7,Xcc grew quickly,and canker symptoms appeared.Plasma membrane(PM)H^(+)-ATPase activation with fusicoccin(FC)acidified the apoplast and upregulated the pathogenesis-related genes(PRs)in the sweet orange leaves.Hence,Xcc colonization and canker development were inhibited.The results of this study revealed that apoplastic acidification is implicated in the resistance of Citron C-05 to Xcc infection and provided insight into the association between pH_(apo)regulation and resistance to bacterial pathogen invasion in plants.展开更多
Citrus canker,caused by Xanthomonas citri subsp.citri(Xcc),is a globally quarantine disease infecting nearly all Citrus cultivars.Citron C-05 has been identified with complete and active resistance to Xcc.However,the ...Citrus canker,caused by Xanthomonas citri subsp.citri(Xcc),is a globally quarantine disease infecting nearly all Citrus cultivars.Citron C-05 has been identified with complete and active resistance to Xcc.However,the mechanism underlying Citron C-05's resistance to Xcc remains elusive.We identified a gene cluster on chromosome 8 of the citrus genome comprising five pathogenesis-related 4-like genes.PR4A was upregulated in Citron C-05 leaves under Xcc infection,exhibiting the highest expression among these PR4-like genes.In addition,PR4A expression was higher in leaves of disease-resistant genotypes than susceptible genotypes under Xcc invasion.Bimolecular fluorescence complementation(BiFC)and Split-Luc assays indicated that CmWRKY75,a positive regulator of PR4A,interacted with pthA4 and upregulated expression of PR4A in Citron C-05 leaves.Regulatory function for the expression of CmPR4A was localized to a 516-nucleotide region upstream of the translational start site,which was designated Pro CmPR4A-P516.Transient overexpression of CmPR4A improved resistance to Xcc in sweet orange,and three transgenic lines of OE-CmPR4A exhibited resistance to Pseudomonas syringae pv.tomato DC3000(Pst DC3000)in Arabidopsis.Furthermore,CmSMU2 was identified through yeast two-hybrid library using CmPR4A as bait,Bi FC and Split-Luc assays further verified their interaction.Transient overexpression of CmSMU2 in sweet orange increased resistance to Xcc.Co-expression of CmSMU2 and CmPR4A enhanced accumulation of reactive oxygen species compared to CmSMU2 or CmPR4A,indicating that they may synergistically enhance resistance to Xcc in citrus.These findings lay the groundwork for a theoretical analysis of the mechanism underlying the resistance of Citron C-05 against citrus canker.展开更多
Orah is a hybrid of ＂Temple＂ tangor and ＂Dancy＂ tangerine, and due to its excellent performance in cultivation in Wuming of Guangxi Province for three years, it has been popularized in Guangxi, Chongqing, Sichuan ...Orah is a hybrid of ＂Temple＂ tangor and ＂Dancy＂ tangerine, and due to its excellent performance in cultivation in Wuming of Guangxi Province for three years, it has been popularized in Guangxi, Chongqing, Sichuan and Yunnan rapidly, with a planting area over 130 000 hm^2. However, there also have been some problems existing in practical production, such as leaf yellowing and canker. The causes for leaf yellowing mainly include the physiologic type and the diseases and pests type. This paper analyzed the causes and put forwards corresponding countermeasures.展开更多
[ 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 endoph...[ 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展开更多
[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 commerc...[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.展开更多
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...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.展开更多
[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） pro...[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.展开更多
Urban trees are subjected to different damaging agents throughout their lifetime. The aims of this study were to identify tree damaging agents, and to obtain a Damage Severity Index (DSI) in order to categorize tree h...Urban trees are subjected to different damaging agents throughout their lifetime. The aims of this study were to identify tree damaging agents, and to obtain a Damage Severity Index (DSI) in order to categorize tree health condition at San Juan de Aragon Park. Each tree was identified at species level in 28 randomly established plots in ten sections of the study area. Up to two types of damage were recorded per tree, based on the FIA (Forest Inventory an Analysis Program) protocol, and a DSI was obtained for each damaged tree considering location of damage, nature of the damaging agent and severity. A total of 753 trees were assessed and 12 species and 27 damaging agents were identified. Cankers, galls, the pepper tree psyllid and the red gum lerp psyllid were the most frequent damaging agents. Australian pine, red gum, Mediterranean cypress, Mexican white cedar, and California pepper were the most affected species. The DSI ranged from 3 to 17 and the average was 7.9. Sections J and H and the species California pepper, Australian pine, and Mexican white cedar presented the highest DSI. The tree population had a moderate health condition, while the aforementioned sections and tree species showed the poorest.展开更多
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 resu...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.展开更多
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 p...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.展开更多
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, manage...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.展开更多
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 togeth...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.展开更多
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,Dick...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.展开更多
The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv （single chain variable fragment） genes, providing the possibility to better u...The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv （single chain variable fragment） genes, providing the possibility to better understand the pathogenesis mechanism via PthA, and developing proper construct for future experimentation to obtain citrus plants resistant to canker disease by transformation and plant antibody techniques. The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti- PthA-NLS McAb, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR （splicing by overlap extension）, and then the ScFv gene was isolated. The recombinant ScFv gene was cloned into pGEM-T and pET32a（＋） vector. The later plasmid was transferred into E. coli BL21 （DE3） and the expression of the recombinant protein was induced. Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6, and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain, and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 kDa of recombinant fusion protein was obtained. In conclusion, we obtained three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS, and the relative ScFv gene was constructed and successfully expressed in E. coli. These results may play an important role in further understanding the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.展开更多
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 cank...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.展开更多
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...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.展开更多
基金Universidade Federal de Uberlandia supported L.Coelho,A.R.T.Nascimento and J.C.Santos。
文摘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.
文摘Fungal pathogens causing poplar canker diseases are cosmopolitan in species,and have a wide range of hosts.These pathogens have diverse anamorphs and their morphology overlaps with each other;Their teleomorphs are hardly discovered in nature.Furthermore,the identification of these pathogens is usually limited by the geography,host and taxonomic knowledge.Therefore,a culture-independent method used to identify determine pathogens is expected to be developed for field diagnostics.Through amplifying,sequencing and analyzing multiplex nucleic acid templates and genetic marked target sequences,a multiplex PCR technique has been established and used to directly detect various environmental samples.In this study, four pathogen strains and environmental samples were amplified using fungal universal primers ITS1/ ITS4 and EF1-728F/EF1-986R by general PCR and multiplex PCR.The amplicons were sequenced,and then aligned in GenBank.The result showed that the multiplex PCR was able to successfully amplify the target gene and identify the pathogens from environmental samples in the condition of an annealing temperature of 55.6℃and primers final concentration of 0.2μmol·L<sup>-1</sup>.The multiplex PCR could amplify the target at the concentration level of approximately lng of genomic DNA.This method would provide a useful tool for the identification of canker pathogens by the multiplex PCR and the high-throughput DNA microarray detection of environmental samples.
基金supported by grants from the National Key Research and Development Program of China(Grant No.2022YFD1400200)the Special Support Plan for High-Level Talent of Shaanxi Province。
文摘Ambient temperature affects the occurrence and prevalence of plant disease.Most bacterial diseases are damaging at high temperatures.However,kiwifruit bacterial canker caused by Pseudomonas syringae pv.actinidiae(Psa)has been found to be prevalent at relatively cool temperatures,and it is unclear how ambient temperature affects the development of kiwifruit bacterial canker.In this study,basal resistance to Psa was suppressed in kiwifruit at cool growth temperature(16℃)compared with at normal temperature(24℃).In addition,RNA sequence analysis and ethylene content assessment indicated that ethylene modulated kiwifruit resistance to Psa at normal growth temperature and that cool temperature inhibited ethylene accumulation and Psa-induced activation of the ethylene signaling pathway in kiwifruit.Virusmediated silencing of the kiwifruit ethylene signaling gene AcEIN2 suppressed kiwifruit resistance to Psa at normal growth temperature.Exogenous application of ethylene inhibitor 1-methylcyclopropene eliminated the difference in kiwifruit resistance to Psa at 16 and 24℃.Exogenous application of ethylene analogues ethephon induced resistance to Psa in kiwifruit.In conclusion,cool temperatures impair basal resistance to Psa by reducing the activation of ethylene biosynthesis and signaling in kiwifruit.The results provide clues for new strategies to control plant diseases in a context of global environmental change.
基金supported by grants from the Provincial Special Project of the Research and Demonstration of High-Efficiency Breeding Technology for Citrus,China(Grant No.2024sfq02)Postgraduate Scientific Research Innovation Project of Hunan Province,China(Grant No.CX20210671).
文摘The p H plays a key role in the growth and colonization of plant pathogens as well as the onset and progression of the symptoms they cause within the host.Plants may quickly alter their apoplastic p H(pH_(apo))to protect themselves against infection.However,pathogens can also alter the p H of their ambient environment to promote their own growth.Citrus canker is a serious plant disease caused by Xanthomonas citri subsp.citri(Xcc).This Gram-negative aerobic rod is usually cultured in Luria-Bertani(LB)medium at p H 7.However,little is known about the changes in p H both in this medium as Xcc grows and in the leaf apoplast in response to Xcc infection and colonization.Moreover,the differences in leaf apoplast p H between Xcc-resistant and Xcc-susceptible citrus genotypes are also unknown.Here,Xcc grew well in liquid LB medium at initial p H6-8 and the pathogen altered the medium p H to 6.8±0.4.Xcc growth declined at p H 5 and was zero at p H 3,4,9,and 10.In susceptible sweet orange infected with Xcc inoculum,canker symptoms were inhibited at p H 3,4,and 10 but did not differ in the range of p H 5-9.As expected,canker symptoms were absent at all inoculum p H in the resistant Citron C-05.For both genotypes,Xcc only grew well in the leaves exposed to p H 5-8 inoculums.At four days post-inoculation(4 dpi),the foliar pH_(apo)of resistant Citron C-05 had rapidly declined from 5.6 to 4.4.At 2 dpi,the p Hapoof susceptible sweet orange had rapidly increased from 5.6 to 6.7,Xcc grew quickly,and canker symptoms appeared.Plasma membrane(PM)H^(+)-ATPase activation with fusicoccin(FC)acidified the apoplast and upregulated the pathogenesis-related genes(PRs)in the sweet orange leaves.Hence,Xcc colonization and canker development were inhibited.The results of this study revealed that apoplastic acidification is implicated in the resistance of Citron C-05 to Xcc infection and provided insight into the association between pH_(apo)regulation and resistance to bacterial pathogen invasion in plants.
基金supported by the National Natural Science Foundation of China(32402500 and 31741110)the Scientific Research Fund of Hunan Provincial Education Department,China(21B0220)。
文摘Citrus canker,caused by Xanthomonas citri subsp.citri(Xcc),is a globally quarantine disease infecting nearly all Citrus cultivars.Citron C-05 has been identified with complete and active resistance to Xcc.However,the mechanism underlying Citron C-05's resistance to Xcc remains elusive.We identified a gene cluster on chromosome 8 of the citrus genome comprising five pathogenesis-related 4-like genes.PR4A was upregulated in Citron C-05 leaves under Xcc infection,exhibiting the highest expression among these PR4-like genes.In addition,PR4A expression was higher in leaves of disease-resistant genotypes than susceptible genotypes under Xcc invasion.Bimolecular fluorescence complementation(BiFC)and Split-Luc assays indicated that CmWRKY75,a positive regulator of PR4A,interacted with pthA4 and upregulated expression of PR4A in Citron C-05 leaves.Regulatory function for the expression of CmPR4A was localized to a 516-nucleotide region upstream of the translational start site,which was designated Pro CmPR4A-P516.Transient overexpression of CmPR4A improved resistance to Xcc in sweet orange,and three transgenic lines of OE-CmPR4A exhibited resistance to Pseudomonas syringae pv.tomato DC3000(Pst DC3000)in Arabidopsis.Furthermore,CmSMU2 was identified through yeast two-hybrid library using CmPR4A as bait,Bi FC and Split-Luc assays further verified their interaction.Transient overexpression of CmSMU2 in sweet orange increased resistance to Xcc.Co-expression of CmSMU2 and CmPR4A enhanced accumulation of reactive oxygen species compared to CmSMU2 or CmPR4A,indicating that they may synergistically enhance resistance to Xcc in citrus.These findings lay the groundwork for a theoretical analysis of the mechanism underlying the resistance of Citron C-05 against citrus canker.
基金Supported by the National Citrus Industry System(CARS-27)Key Project of science and Technology from the Agriculture Department of Guangxi Province(201504,201307)+4 种基金Scientific and Technological Development Project of Nanning City(NC20152302,20142062)Scientific and Technological Project of Wuming County(201501217)Fund of Guangxi Academy of Agricultural Sciences(NCZ2015010,GNK2015JM20,GNK2014YZ36,GNK2015YT51,GNK2016YM47)Central Financial Project of Agriculture Technology Extension in 2015Project for the Construction of Good Fruit Variety Breeding Center in Guangxi~~
文摘Orah is a hybrid of ＂Temple＂ tangor and ＂Dancy＂ tangerine, and due to its excellent performance in cultivation in Wuming of Guangxi Province for three years, it has been popularized in Guangxi, Chongqing, Sichuan and Yunnan rapidly, with a planting area over 130 000 hm^2. However, there also have been some problems existing in practical production, such as leaf yellowing and canker. The causes for leaf yellowing mainly include the physiologic type and the diseases and pests type. This paper analyzed the causes and put forwards corresponding countermeasures.
基金Supported by"Changjiang Scholars and Innovative Research Team"SupportPlan of Ministry of Education(200558)Research Fund for the DoctoralProgram of Three Gorges University(KJ2008B015)~~
文摘[ 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
基金Supported by National Modern Industrial Technology System of Apple(CARS-28)National Natural Science Foundation of China(31000891)~~
文摘[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.
基金National Foundation of Ninth Five-Year Plan (No. 96-005-04-01-03).
文摘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.
基金Supported by National Natural Science Foundation of China(30571489)Shandong Agricultural University Youth Science and Technology Innovation Fund Projects(23406)~~
文摘[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.
文摘Urban trees are subjected to different damaging agents throughout their lifetime. The aims of this study were to identify tree damaging agents, and to obtain a Damage Severity Index (DSI) in order to categorize tree health condition at San Juan de Aragon Park. Each tree was identified at species level in 28 randomly established plots in ten sections of the study area. Up to two types of damage were recorded per tree, based on the FIA (Forest Inventory an Analysis Program) protocol, and a DSI was obtained for each damaged tree considering location of damage, nature of the damaging agent and severity. A total of 753 trees were assessed and 12 species and 27 damaging agents were identified. Cankers, galls, the pepper tree psyllid and the red gum lerp psyllid were the most frequent damaging agents. Australian pine, red gum, Mediterranean cypress, Mexican white cedar, and California pepper were the most affected species. The DSI ranged from 3 to 17 and the average was 7.9. Sections J and H and the species California pepper, Australian pine, and Mexican white cedar presented the highest DSI. The tree population had a moderate health condition, while the aforementioned sections and tree species showed the poorest.
基金This research was supported by National Natural Science Foundation of China (Grant No. 30100143)
文摘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.
基金the National Key Research and Development Program of China(2018YFD1000300,to S.C.)the National Natural Sciences Foundation of China(31972393,to X.Z.)+2 种基金the Natural Science Foundation Project of Chongqing City(cstc2019jcyj-bshX0024 to Q.L.)the Fundamental Research Funds for the Central Universities(XDJK2019C027,to Q.L.)the Earmarked Fund for China Agriculture Research System(CARS-26,to S.C.).
文摘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.
基金supported by the twelfth five-year science and technology support program of China(2012BAD19B0801)
文摘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.
基金financially supported by the National HighTech R&D Program of China (863, 2011AA100205)the National Natural Science Foundation of China (31125024)
文摘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.
基金supported by the 111 International Cooperation Grant 2.0(BP0719029)to Nanjing Agricultural University,China,from the Chinese government and Canadian Interdepartmental funding of Genomics Research and Development Initiatives(GRDI)。
文摘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.
基金supported by the Scientific and Technical Project of Hunan Province,China(04NK1005)
文摘The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv （single chain variable fragment） genes, providing the possibility to better understand the pathogenesis mechanism via PthA, and developing proper construct for future experimentation to obtain citrus plants resistant to canker disease by transformation and plant antibody techniques. The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti- PthA-NLS McAb, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR （splicing by overlap extension）, and then the ScFv gene was isolated. The recombinant ScFv gene was cloned into pGEM-T and pET32a（＋） vector. The later plasmid was transferred into E. coli BL21 （DE3） and the expression of the recombinant protein was induced. Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6, and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain, and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 kDa of recombinant fusion protein was obtained. In conclusion, we obtained three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS, and the relative ScFv gene was constructed and successfully expressed in E. coli. These results may play an important role in further understanding the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.
文摘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.
基金supported by the National Key Research and Development Program of China(2021YFD1400800,2022YFD1400200)the National Natural Sciences Foundation of China(31972393)+1 种基金the Fundamental Research Funds for the Central Universities(SWU-XDJH202308,XDJK2018B016)the Earmarked Fund for China Agriculture Research System(CARS-27).
文摘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.
