Citrus Canker. Nian Wang 11/20/2017

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1 Citrus Canker Nian Wang 11/20/2017

2 Citrus canker disease and pathogen Xanthomonas citri subsp. citri fruit drop, defoliation, yield losses 5-30%, fruit marketability quarantine restrictions Gottwald 2002

3 Host range Host range: Affects plants of Rutaceae family, commonly known as the citrus family, primarily Citrus spp., Fortunella spp., and Poncirus spp., world-wide. Quarantined pathogen in many countries. Economically important hosts are cultivated orange, grapefruit, lime, lemon, pomelo and citrus rootstock (Brunings and Gabriel 2003).

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5 Historical strain types A-strain (Xanthomonas citri subsp. citri; X. citri; X. campestris pv. citri; X. axonopodis pv. citri): The most aggressive and native to Asia. B-strain (X. axonopodis pv. aurantifolii ): Argentina, Paraguay and Uruguay. C-strain (X. axonopodis pv. aurantifolii ): Brazil. A W : Wellington, Florida A*: Asiatic A* group from south-west Asia E strain: Bacterial spot strain (misdiagnosis), nursery strain

6 Characteristics of xanthomonads Classification: Kingdom: Bacteria Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Xanthomonadales Family: Xanthomonadaceae Genus: Xanthomonas Gram-negative Aerobic Rods with a single polar flagellum Mucoid Extracellular polysaccharide: Xanthan gum

7 Characteristics of xanthomonads The unique yellow pigment-xanthomonadins (Absorption peak between 443 and 446 nm). Most Xanthomonas bacteria produce yellow, membrane bound, brominated aryl-polyene pigments referred to as xanthomonadins. Xanthomonadins are unique to Xanthomonas bacteria and serve as useful chemotaxonomic and diagnostic markers. Protection against photobiological damage With methods of artificial infection, xanthomonadindeficient strains were not affected in pathogenicity, symptomatology, or in planta growth. Thus, the xanthomonadins apparently are not important to the pathogen after infection of the host plant.

8 The aggressive citrus canker pathogen Xanthomonas citri subsp. citri 1910 Canker identified on imported seedlings from Japan in Florida for first time Canker eradicated New detection in Manatee County 1994 Eradication declared, remerged in Canker detected for a third time near Miami international Airport. Susan Salisbury Tim Gottwald 1900-ft radius Eradication zone

9 Xanthomonas axonopodis pv. citrus is limited in the intercellular spaces

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11 Citrus leafminer facilitates the infection of X. citri subsp. citri Larva Gottwald et al Asian citrus leafminer, a pest first detected in 1993 in Florida.

12 Hurricanes and spread of citrus canker 8/13/04 Charley 9/5/04 Frances 9/25/04 Jeanne 2006 January 10 USDA withdraws funds for eradication. All tree removal ceased. Recent spread. Learn to live with citrus canker. Irey et al. 2006

13 Management for Prevention and Control of Citrus Canker Quarantine measures to prevent the introduction and establishment of X. citri subsp. citri. Eradication program Cultivar selection. Resistant cultivars. Mandarins in Asia. Marketability of certain varieties in USA. Exclusion/Sanitation procedures. Decontamination of personnel and equipment to reduce the risk of spread. Cultural practices: windbreaks, pruning or defoliation of diseased summer and autumn shoots. Chemical control: copper-based bactericides. The toxic effect of copper against X. citri takes place only at the surface without any internal and/or systemic action. Problem with copper-based bactericides.

14 Disadvantages of long-term use of copper based bactericides Resistance to copper in xanthomonad populations Accumulation of copper metal in soils with potential phytotoxic and environmental effects.

15 Copper resistant strains The development of Cu r strains of X. citri subsp. citri has been reported only in Argentina in 1994 from a citrus grove located in the province of Corrientes which showed a lack of response to the numerous copper sprays. Copper resistance has also been identified in X. axonopodis pv. citrumelo, the causal agent of citrus bacterial spot disease. Most copper resistance genes are plasmid borne, some are located on the chromosome. copabcd

16 Virulence mechanism of X. citri subsp. citri

17 Type III secretion system and T3SS effectors are critical for causing citrus canker symptom ptha4 Büttner & He 2010 W M Concentration (CFU cm 2 ) Xac306 (WT) 263A6 (XAC0396) Days post inoculation W M cell density (CFU/cm 2 ) XAC306 (WT) 234B2 (XAC0405) DPI (days post inoculation) W cell density (CFU/cm 2 ) M XAC306 (WT) 308F7 (XAC0412) DPI (days post inoculation)

18 PthA is the pathogenicity gene of X. citri Yan and Wang 2012 MPMI PthA is responsible for hypertrophy and hyperplasia, which are important for bacterial growth and bacterial release from citrus canker lesions for further transmission.

19 (A) Normal citrus leaf. (B) citrus leaf following particle bombardment with ptha under control of a plant promoter. (C) citrus leaf following artificial inoculation with X. citri. Note the loss of free space in the spongy mesophyll region, the necrosis (N) of the abaxial epidermal layer, and X. citri bacteria (B) oozing from the artificial inoculation. (Duan et al., 1999.)

20 PthA is a transcription activator-like (TAL) effector which induces the expression of canker susceptibility gene CsLOB1 PthA4 Repeats (17.5) NLS NLS NLS AD RVDs EBE PthA L T P E Q V V A I A S N IG G K Q A L E T V Q R L L P V L C Q A H G NI NG NI NI NI HD HD NG HD NG NG NG NG NS HD HD NG NG T A T A A A C C T C T T T T A C C T T 34aa EBE PthA4 CsLOB1

21 Generating canker resistant citrus varieties by modifying S gene CsLOB1 using SpCas9/sgRNA Jia et al. 2016

22 Nursery strain-citrus bacterial spot Opportunistic leaf spotting xanthomonads that did not cause cankers or affect the fruit of mature trees were misdiagnosed in Florida in 1984 as a another "form" of citrus canker, E-strain. Citrus bacterial leaf spot", or "nursery leaf spot" caused by X. campestris pv. citrumelo (X. campestris pv. citrumelo; X. alfalfae pv. citrumelonis) was found in Florida in 1984 and reported to be a new "form" of citrus canker. Lesions on leaves were not erumpent (raised) as in the case of the Asiatic strain but rather mostly flat and sometimes sunken. It was classified as another strain of X. axonopodis pv. citri, necessitating regulatory procedures.

23 Comparison between citrus bacterial spot and citrus canker Citrus bacterial spot disease Limited primarily to trifoliate orange, its hybrids, e.g. Swingle citrumelo. Occurs exclusively in nurseries Citrus canker Broad host range, including most commercial citrus Endemic in Florida in most citrus-producing areas

24 Nursery strain Strains are closely related to pathovar alfalfae. Thus, they are not X. c. pv. citri but genetically distinct and, therefore, called X. c. pv. citrumelo A USDA quarantine and grower compensation program was the result, despite the fact that cankers were never observed, and mature trees in groves were not affected. Eradication of bacterial spot disease, resulting in the destruction of 20 million citrus plants at the cost of $94 million.

25 Xanthomonas oryzae pv. oryzae KACC Xanthomonas oryzae pv. oryzae PXO99A Xanthomonas oryzae pv. oryzae MAFF Xanthomonas campestris pv. vesicatoria str Xanthomonas axonopodis pv. citrumelo str. F1 Xanthomonas axonopodis pv. citri str Xanthomonas campestris pv. campestris str. ATCC Xanthomonas campestris pv. campestris str Xanthomonas campestris pv. campestris str. B Xylella fastidiosa Temecula1 79 Xylella fastidiosa M12 Xylella fastidiosa 9a5c Xanthomonas albilineans GPE PC73 Stenotrophomonas maltophilia R551-3 Stenotrophomonas maltophilia k279a Psuedomonas syringae pv. tomato str. DC3000 Psuedomonas syringae pv. syringae B728a Psuedomonas aeruginosa PA7 Ralstonia solanacearum str. GMI0 Ralstonia solanacearum str. PSI Burkholderia mallei NCTC 10247