A single amino acid substitution in PthA of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis> altering canker formation on grapefruit leaves

The typical citrus canker lesions produced by Xanthomonas axonopodis pv. citri are erumpent, callus-like, with water-soaked margins. Three novel atypical symptom-producing variants of X. axonopodis pv. citri were described recently in Taiwan. Only the variant designated as A^f type produces typical erumpent canker lesions on Mexican lime (Citrus aurantifolia) but induces flat necrotic with water-soaked margin lesions on grapefruit leaves (C. paradisi). Two homologous pthA were cloned and characterized from strains XW19 (a typical canker lesion producing strain) and XW47 (a strain of A^f type). The pthA homolog from XW19 was transformed into XW47. The transformant of XW47 induced typical erumpent canker lesions on grapefruit leaves. Sequence analyses of transformants XW19 and XW47 revealed over 99% homology in nucleotide and deduced amino acid sequences compared with pthA homologs deposited in GenBank. The amino acid residues located at positions 49, 286, 742 and 767 of PthA were different between XW47 and XW19. The PthA mutants with a single amino acid substitution at each of these four positions were constructed by site-directed mutagenesis. Modified PthA (S286P) from XW47 in transformant 47SP induced erumpent canker lesions on grapefruit leaves, whereas another modified PthA (P286S) from XW19 in transformant 47PS only induced flat necrotic lesions. These results suggested that a single amino acid substitution from either serine to proline or proline to serine at position 286 of PthA can alter canker formation by X. axonopodis pv. citri on grapefruit leaves.     

Functional analysis of the 3′ end of avrBs3/pthA genes from two Xanthomonas species

The phytopathogens Xanthomonas oryzae pathovar (pv.) oryzae and Xanthomonas axonopodis pv. citri each contain several avrBs3/pthA family genes. Structural features of these genes important for avirulence and/or virulence functions include a central region of multiple direct repeats and three nuclear localization signals (NLSs) and an acidic activation domain (AAD) at the 3′ end. To identify other regions critical to function in the 3′ ends of these genes, we constructed several chimeras using apl1 and apl2 from X. axonopodis pv. citri and avrXa10 and avrXa7 from X. oryzae pv. oryzae and evaluated their functions by inoculation to citrus and rice. The apl1 and avrXa7 genes are major virulence determinants in citrus and rice, respectively, while the contributions of apl2 and avrXa10 to virulence are negligible or not measurable. Constructs that contained a 417 bp HincII-SphI fragment from the 3′ end of apl1 in combination with the repeats from avrXa7, avrXa10, and apl1 caused a canker phenotype on citrus. Interchange of the HincII-SphI fragment between avrXa7 and avrXa10 abolishes avrXa7 avirulence function and reduces its virulence but it does not affect avrXa10 avirulence function in rice. avrXa7 caused a hypersensitive response (HR) in citrus and replacement of it's 3′ end with that of apl1 resulted in loss of canker and induction of HR. Thus, the HincII-SphI fragment of the avrBs3/pthA gene family is important for avirulence and virulence functions in two different plant species, Oryza sativa and Citrus natsudaidai HAYATA.     

Susceptibility to citrus canker caused by <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis> depends on the nuclear genome of the host plant

Susceptibility to Xanthomonas axonopodis pv. citri of a citrus cybrid, in which the nuclear and cytoplasmic genomes were derived from Citrus sinensis and C. unshiu, respectively, was evaluated. Bacterial growth in the leaves of the cybrid was similar to that in C. sinensis after pin-prick inoculation throughout the experiment, whereas growth was greater than that in C. unshiu from 8 days after inoculation. Lesion expansion and pustules that later developed from the lesions on the cybrid and on C. sinensis also appeared to be greater than those on C. unshiu. The incidence of citrus canker disease caused by the bacteria on the cybrid trees was in the field was equivalent to that on C. sinensis but was severer than on C. unshiu. These results indicate that the nuclear genome of the cybrid plays a critical role in susceptibility to citrus canker disease. However, the pathogenicity gene pthA of the bacteria is not likely to be involved in the difference in susceptibility to the bacteria between C. unshiu and C. sinensis because their susceptibility to a pthA-deficient mutant of the bacteria also differs.     

Over-expression of the citrus gene CtNH1 confers resistance to bacterial canker disease

Citrus canker is a devastating disease, caused by Xanthomonas axonopodis pv. citri (Xac). It is well established that the NPR1 gene plays a pivotal role in systemic acquired resistance (SAR) in Arabidopsis. In this study, we report the isolation and characterization of an NPR1 homolog from citrus, namely Citrus NPR1 homolog 1 (CtNH1). Sequence alignment and phylogenetic analysis indicate that CtNH1 is closely-related to the Arabidopsis NPR1 gene and its orthologs from rice, grapevine, and cacao. When over-expressed in citrus, CtNH1 confers resistance to Xac and leads to constitutive expression of the pathogenesis-related (PR) gene chitinase 1 (Chi1), suggesting that CtNH1 is orthologous to NPR1.     

Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot

A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10⁴, 10⁵, or 10⁸ cfu ml⁻¹ were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10⁸ cfu ml⁻¹), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10⁵ cfu ml⁻¹, populations 14 dpi were <10⁴ per inoculation site. In canker-susceptible Citrus spp. (‘Duncan’ grapefruit and ‘Rough’ lemon), water-soaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10⁵ cfu ml⁻¹ by 14 dpi. In citrus bacterial spot-susceptible citrus (‘Swingle’ citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10⁵ cfu ml⁻¹ were comparable to Xcc in susceptible hosts 14 dpi (>10⁵). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.     

Soil application of imidacloprid and related SAR-inducing compounds produces effective and persistent control of citrus canker

Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced season-long control of citrus canker caused by Xanthomonas citri sbsp. citri. Imidacloprid is a neo-nicotinoid that breaks down in planta into 6-chloronicotinic acid, a compound closely related to the systemic acquired resistance (SAR) inducer isonicotinic acid. Potted Swingle citrumelo seedlings (Citrus paradisi × Poncirus trifoliata) were treated with imidacloprid and the SAR inducers, isonicotinic acid, and acibenzolar-s-methyl as soil drenches or with acibenzolar-s-methyl as a foliar spray 1week prior to inoculation of immature leaves with X. citri sbsp. citri. Seedlings were re-inoculated four times over a 24-week period. SAR induction was confirmed by expression of the PR-2 gene (β-1,3 glucanase). Soil drenches of imidacloprid, isonicotinic acid, and acibenzolar-s-methyl induced a high and persistent up-regulation of PR-2 gene expression and reduced the number of canker lesions for up to 24 weeks compared to 4 weeks for foliar acibenzolar-s-methyl. Soil applied inducers of SAR reduced canker lesions up to 70% compared with the untreated inoculated plants. Lesions on leaves were small, necrotic, and flat compared to pustular lesions on inoculated untreated plants. Populations of X. citri sbsp. citri per leaf were reduced 1–3 log units in soil-treated plants compared to inoculated untreated plants.     

Unstable green fluorescent protein for study of Xanthomonas citri subsp. citri survival on citrus

Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus bacterial canker, an important disease for the citrus industry. Studies of Xac survival in environments outside of the lesion performed in the past may have underestimated the viable population because the recovery was based on the ability of the bacterium to grow on culture media. This study monitored survival of Xac that express green fluorescent protein (GFP) in two different forms: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Transformed strains of Xac were verified to be stable in their expression of GFP and to show no differences in virulence and fitness compared to wild type strains. Evaluation of protein stability confirmed that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal microscopy was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. Aggregation of viable bacteria in biofilms confirms their role in pathogen survival outside of lesions and protection from bactericide treatments in the field or in the fruit disinfection process.     

Hsp70 and Hsp90 expression in citrus and pepper plants in response to <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

Hsp70 and Hsp90 expression in response to high and low temperatures was studied in orange, the host plant of Xanthomonas axonopodis pv. citri and in a non-host resistant plant, pepper. As expected in both plants, the expression of these chaperones was induced at high temperatures while at cold temperatures the response was chaperone and plant-dependent. Expression of Hsp70 and Hsp90 was analysed during citrus canker and no changes in their levels could be observed whereas pepper plants infiltrated with the phytopathogen showed an increase in the levels of both chaperones. These results suggest that no changes in Hsp70 and Hsp90 expression are necessary during the disease while they are increased in non-host resistance.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.     

Bacterial brown spot on Avena storigosa Schereb. caused by Pseudomonas syringae pv. alisalensis

Avena storigosa Schereb. (bristle oat) is used as a green manure in crop rotations and as an antagonist of nematodes in Nagano Prefecture, Japan. In 2011, necrotic, brown, water-soaked lesions were observed on young bristle oat plants. A pathogenic bacterium was isolated from symptomatic leaves of infected plants and produced the same symptoms after inoculation. Bacteriological properties of the bacterial isolates from bristle oat matched those of Pseudomonas syringae pathovars. The host range of the bristle oat isolates was identical to that of P. syringae pv. alisalensis. This is the first report of bristle oat disease caused by P. syringae pv. alisalensis.     

Consequences of the taxonomic revision of Xanthomonas axonopodis pv. dieffenbachiae and of the analysis of its associated pathogenicity for the recommendation of regulation (EPPO A2 List)

Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of bacterial blight of Araceae (aroids), is a regulated pest in several countries and is included in the EPPO A2 List. Reference strains of Xanthomonas axonopodis pv. dieffenbachiae have recently been reclassified into the species Xanthomonas phaseoli, Xanthomonas citri and Xanthomonas euvesicatoria on the basis of different features, including multilocus sequence analysis, average nucleotide identity and homology in DNA–DNA hybridization analyses. Based on pathogenicity tests, Constantin et al. (2017) proposed naming the pathogens on aroids as X. phaseoli pv. dieffenbachiae, X. phaseoli pv. syngonii and X. citri pv. aracearum. Recommendations are made on how to deal with these changes for the group of pathogenic bacteria for Araceae. The name Xanthomonas axonopodis pv. dieffenbachiae on the EPPO List should be adjusted to the names proposed in the taxonomic study by Constantin et al. (2016). The current EPPO Diagnostic Standard is directed at strains pathogenic on Anthurium. They mainly belong to X. phaseoli pv. dieffenbachiae, but some also to X. citri pv. aracearum that are not detected by the EPPO Diagnostic Standard. Xanthomonas phaseoli pv. syngonii strains are also aggressive, but with a host range restricted to Syngonium. The pathogenicity specific to aroids of the bacterial isolates reclassified as Xanthomonas euvesicatoria was not confirmed and no pathovar epithet has been retained for these strains.     

Role of phaseolotoxin production by Pseudomonas syringae pv. actinidiae in the formation of halo lesions of kiwifruit canker disease

Pseudomonas syringae pv. actinidiae, the causal bacterium of kiwifruit canker, induces the formation of chlorotic halo lesions on infected leaves and inhibits the growth of Escherichia coli. The inhibition ofE. coli growth was shown to be reversed by L -arginine or L -citrulline, but not by L -glutamine, suggesting that the pathogen produces a toxin similar to phaseolotoxin, which inhibits ornithine carbamoyltransferase. The toxin was purified from culture broth of P. syringae pv. actinidiae strain Kw11, and was shown by nuclear magnetic resonance to be identical to phaseolotoxin. Assays based on inhibition of E. coli growth and on amplification of a phaseolotoxin fatty acid desaturase gene (ptx) fragment revealed that, among the plant pathogenic bacteria examined, the production of phaseolotoxin is restricted to strains of P. syringae pv. phaseolicola and pv.actinidiae . A non-toxigenic mutant of strain Kw11 generated by disruption of the ptx gene induced the formation of necrotic lesions on kiwifruit leaves; however, the lesions were not surrounded by a chlorotic halo as were those induced by the parent strain. The growth rate of the non-toxigenic mutant in leaf tissue was similar to that of the parent strain. These results suggest that phaseolotoxin production contributes to the formation of chlorotic halo lesions in kiwifruit canker but is not required for multiplication of the pathogenic bacterium during infection.     

Genomic sequencing globally identifies functional genes and potential virulence-related effectors of Xanthomonas axonopodis pv. malvacearum

Highly virulent strains (HVS) of Xanthomonas axonopodis pathovar (pv.) malvacearum (Xam) infect all commercial cultivars of cotton, including the long-established “immune” cultivar 101-102B. Here, we present high-quality draft sequences of a highly virulent Xam strain (GSPB2388) from Sudan, and a strain of race 18 (GSPB1386) from Nicaragua, using Illumina/Solexa paired-end sequencing. The short sequence reads were assembled into 61 scaffolds for GSPB2388 (N50 of 164 kb) and 127 scaffolds for GSPB1386 (N50 of 100 kb), with draft maps of roughly 5 Mb, which contain 4,665 and 4,520 protein-coding genes, respectively. Through gene annotation and comparisons with plant pathogen proteins, 181 and 178 potential virulence-related genes, including genes encoding a major group of type III effectors, were identified from GSPB2388 and GSPB1386, respectively. The differential effectors and sequence diversity between the HVS and race 18 may enable the identification of key factors that contribute to the high virulence of HVS. Additionally, the average nucleotide identity (ANI) between Xam and Xanthomonas axonopodis pv. citri is 98.4 %, suggesting that these strains belong to the same species.     

Effect of the duration of inoculum exposure on development of citrus canker symptoms on seedlings of Swingle citrumelo

Citrus canker (Xanthomonas citri subsp. citri, Xcc) is one of the most serious diseases citrus in Florida, and elsewhere in the world. The disease causes yield loss and some fresh fruit trade restrictions may apply. Cultural management techniques such as windbreaks may work by not only reducing wind speed, but also reducing the period of exposure of susceptible foliage or fruit to those wind speeds that support infection from incoming inoculum. To investigate the effect of exposure period to inoculum of Xcc, seedlings of canker-susceptible Swingle citrumelo were exposed to sprayed inoculum for increasing periods at different wind speeds. The incidence and severity of citrus canker was assessed. In three experiments the incidence and severity of citrus canker most often increased with longer periods of exposure to inoculum, especially so at wind speeds of ≥16 m/s compared to wind speeds of ≤5 m/s (wind speed also increased disease incidence and severity). Regression analysis demonstrated relationships between period of exposure to inoculum and the percent infected leaves per plant, the number of lesions per plant, the number of lesions per infected leaf, and for the percent of infected leaves with lesions on the petioles at wind speeds of ≥16 m/s (R²?=?0.16–0.72). Due to the effect of inoculum exposure period and wind speed, attempts should be made to minimize exposure of canker-susceptible citrus when wind speed is highest and inoculum is available. Windbreaks should help minimize periods of exposure to splashed inoculum in high winds.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

HrpG regulates type II secretory proteins in Xanthomonas axonopodis pv. citri

HrpG, a two-component response regulator-like protein, is a key regulator of the type III secretion system (T3SS) in Xanthomonas spp. In X. campestris pv. vesicatoria, HrpG with a single amino acid substitution (HrpG*) gains the ability to induce the expression of T3SS-related genes even under nutrient-rich conditions. In this study, we investigated the role of HrpG in the synthesis of the secretory protein using HrpG* in strain NA-1 of X. axonopodis pv. citri (Xac NA-1), a causal agent of citrus canker. Eleven proteins secreted via a type II secretion system (T2SS) were induced by HrpG*. In proteomic analyses, six of the 11 proteins were identified as extracellular enzymes, and the others as a fimbrial biogenesis-related protein, a type IV-related protein, two hypothetical proteins, and a conserved hypothetical protein. Further analysis of these proteins revealed that the genes coding all 11 proteins were upregulated by HrpG*, even though they had different expression patterns for HrpXct-dependency. The data indicated that HrpG, a key regulator of T3SS, also acts as a positive regulator of certain proteins secreted via a T2SS in Xac NA-1.     

A functional XopAG homologue in Xanthomonas fuscans pv. aurantifolii strain C limits host range

Citrus canker is caused by two Xanthomonas species, Xanthomonas citri, which has become the primary pathogen where citrus canker occurs (type A citrus canker, Xc‐A), and X. fuscans pv. aurantifolii (Xfa), which consists of strains B and C. The B strain is less pathogenic than the A strain, but produces symptoms in all citrus species. The C‐type cankers only infect Key lime (Citrus aurantifolia) and produce a hypersensitive reaction (HR) in grapefruit (Citrus paradisi) leaves. An avirulence gene, avrGf2, was identified in a C strain that was responsible for the HR in grapefruit. AvrGf2 is a member of XopAG effector family and shares 45% identity at amino acid level with another member of the same family, AvrGf1 from strain Xc‐A^w, which was previously shown to elicit an HR in grapefruit. AvrGf2 shares sequence identity with other XopAG effectors present in Xanthomonas vasculorum, Xanthomonas campestris pv. musacearum and Pseudomonas syringae pv. tomato. Mutagenesis of avrGf2 in C strain resulted in a compatible reaction in grapefruit. There was no observable effect on virulence when Xc‐A transconjugants containing either avirulence gene were inoculated on Key lime. Expression of avrGf1 or avrGf2 in Xc‐A resulted in a similar phenotype following infiltration into grapefruit leaves, although the avrGf2 transconjugant elicited a faster HR and lower populations than the transconjugant containing avrGf1. Also, it was shown that all Xfa‐B strains tested contain a transposon in avrGf2 that helps to explain the differences in host range between B and C strains.