Over-expression of the Arabidopsis <Emphasis Type="BoldItalic">NPR1</Emphasis> gene in citrus increases resistance to citrus canker

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc), is a serious leaf and fruit spotting disease affecting many important citrus cultivars including grapefruit and certain sweet oranges. Currently, efficacious and economical disease control measures for highly susceptible citrus cultivars are lacking. Development of commercial cultivars with greater field resistance to citrus canker is the optimum strategy for effective disease management. In this study, we generated transgenic ‘Duncan’ grapefruit (DG) and ‘Hamlin’ sweet orange (Ham) expressing the Arabidopsis NPR1 gene (AtNPR1), which is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR). Our results indicate that over-expression of AtNPR1 in citrus increases resistance to citrus canker and that the resistance is related with the expression levels of AtNPR1 in the transgenic plants. The line (DG 42-2) with the highest expression level of AtNPR1 was also the most resistant, which developed significant fewer lesions accompanied by a ten-fold reduction in Xcc population. The lesions developed on DG 42-2 were smaller and darker than those on the control and lacked callus formation. These lesion phenotypes resemble those on canker resistant kumquats and canker susceptible citrus trees treated with SAR-inducing compounds. Therefore, over-expression of AtNPR1 in citrus is a promising approach for development of more resistant cultivars to citrus canker.     

Inverse effects of Arabidopsis NPR1 gene on fusarium seedling blight and fusarium head blight in transgenic wheat

In this study, the Arabidopsis thaliana NPR1 (non‐expressor of PR genes) gene was integrated into an elite wheat cultivar, and the response of the transgenic wheat expressing NPR1 to inoculation with Fusarium asiaticum was analysed. With seedling inoculation, the transgenic lines showed significantly increased fusarium seedling blight (FSB) susceptibility, whereas floret inoculation resulted in enhanced fusarium head blight (FHB) resistance. Quantitative real‐time PCR revealed that expression of two defence genes, PR3 and PR5, was associated with susceptible reactions to FSB and FHB, whereas the PR1 gene was activated in resistance responses. This inverse modulation by the constitutively expressed NPR1 gene suggests that NPR1 has a bifunctional role in regulating defence responses in plants. Therefore, it is unsuitable for improving overall resistance to FSB and FHB in wheat.     

Involvement of OsNPR1/NH1 in rice basal resistance to blast fungus Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a major threat to worldwide rice production. Plant basal resistance is activated by virulent pathogens in susceptible host plants. OsNPR1/NH1, a rice homolog of NPR1 that is the key regulator of systemic acquired resistance in Arabidopsis thaliana, was shown to be involved in the resistance of rice to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae and benzothiadiazole (BTH)-induced blast resistance. However, the role of OsNPR1/NH1 in rice basal resistance to blast fungus M. oryzae remains uncertain. In this study, the OsNPR1 gene was isolated and identified from rice cultivar Gui99. Transgenic Gui99 rice plants harbouring OsNPR1-RNAi were generated, and the OsNPR1-RNAi plants were significantly more susceptible to M. oryzae infection. Northern hybridization analysis showed that the expression of pathogenesis-related (PR) genes, such as PR-1a, PBZ1, CHI, GLU, and PAL, was significantly suppressed in the OsNPR1-RNAi plants. Consistently, overexpression of OsNPR1 in rice cultivars Gui99 and TP309 conferred significantly enhanced resistance to M. oryzae and increased expression of the above-mentioned PR genes. These results revealed that OsNPR1 is involved in rice basal resistance to the blast pathogen M. oryzae, thus providing new insights into the role of OsNPR1 in rice disease resistance.     

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.     

A pthA homolog from a variant of Xanthomonas axonopodis pv. citri enhances virulence without inducing canker symptom

Three novel atypical symptom-producing types of Xanthomonas axonopodis pv. citri were described recently. The variants designated as Xac-A^p type produce large flat necrotic with water-soaked margin lesions instead of canker lesions on leaves of Mexican lime (Citrus aurantifolia) and grapefruit (C. paradisi). Random mutagenesis using transposon Tn5 in Xac-A^p type strain XL38 was used to isolate a virulence-deficient mutant T38, which induced small flat necrotic with water-soaked margin lesions on leaves. PthAp being inactivated by the transposon was cloned and characterized. Our result demonstrated that pthAp is responsible for XL38 to cause large flat necrotic with water-soaked margin lesions and bacterial multiplication but fails to confer the ability of inducing canker lesions. Sequence analysis reveled pthAp is a new member belong to the pthA homologs. The sequence is almost identical to the other homologs except for the number of direct tandem repeats in the central region of the gene. The pthAp contained an intact promoter and a full-length reading frame but with 18.5 direct repeats. Moreover, the amino acid at position 3rd in 1st, 2nd, 3rd repeats and at 13th position in 11th, 12th, 13th, 14th repeats were varied. Mutant T38 is fully complemented by pthA carried 17.5 direct repeats for the pathogenicity of X. axonopodis pv. citri to elicit canker lesions. The pthAp affected in planta bacterial growth and lesions in size but no ability of inducing canker symptom. These results suggested that pthAp is a new distinct virulence effector responsible for the enhancement of virulence.     

Widespread occurrence of “Candidatus liberibacter africanus subspecies capensis” in Calodendrum capense in South Africa

Recent studies in citrus orchards confirmed that Citrus Greening, a heat sensitive citrus disease, similar to Huanglongbing (HLB), is associated with the presence of ??Candidatus Liberibacter africanus?? (Laf) in South Africa. Neither ??Candidatus Liberibacter asiaticus?? (Las), associated with HLB, ??Candidatus Liberibacter americanus??, nor ??Candidatus Liberibacter africanus ssp. capensis?? (LafC), previously detected in the Western Cape, South Africa on an indigenous Rutaceous species, Calodendrum capense (L. f.) Thunb. (Cape Chestnut), were detected in citrus. The current study aims to determine the potential role of C. capense in the epidemiology of Citrus Greening in South Africa and whether LafC poses a risk to citriculture. A total of 278?C. capense samples were collected throughout South Africa and tested for Liberibacters using real-time PCR. While LafC was found in 100 samples, distributed from all areas where collected, no evidence of Laf infection in any sample was found . The identity of the LafC present was confirmed by sequencing the amplicon derived from conventional PCR of the ?-operon of the ribosomal protein gene region of the first 17 infected trees found and of a representative sample from each district. The Liberibacter status of 44?C. capense and 272 citrus (Midnight Valencia) trees growing in close proximity to each other for over 15?years was determined. Out of 44?C. capense specimens, 43 were infected with LafC, but none of the citrus trees were infected with LafC. Based on the results of this it appears that natural spread of LafC to citrus does not occur.     

Identification and characterization of known and novel viroid variants in the Greek national citrus germplasm collection: threats to the industry

Seventy four percent of the budwood tree sources samples from the Greek national citrus germplasm foundation collection were positive for one or more viroids. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the two potentially damaging viroids for the Greek citriculture, especially after transitioning to Citrus tristeza virus resistant/tolerant rootstocks and scions, were detected along with Citrus bark cracking viroid, Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). All samples tested negative for Citrus viroid V (CVd-V), CVd-VI and CVd-I-LSS (CBLVd variant). An HSVd isolate related to the non-cachexia variant contained two critical cachexia-related nucleotide changes, while two more isolates were unique among the previously reported HSVds. Unusual CDVd isolates with altered RNA secondary structure were identified in trees additionally co-infected with CEVd and HSVd. Budwood sources that had previously undergone therapy tested negative for all targeted viroids, suggesting that budwood sources in Greece can be protected against graft-transmissible pathogens, even under severe inoculum pressure. Therapied and tested citrus propagative material requires a comprehensive program not available currently in Greece, involving regulators, scientists, and the private sector, for the establishment and successful operation of a national citrus germplasm collection.     

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.     

Reaction of transgenic Citrus sinensis plants to Citrus tristeza virus infection by Toxoptera citricida

Transgenic Citrus sinensis ‘Hamlin’ and ‘Valencia’ plants containing Citrus tristeza virus (CTV)-derived sequences were propagated and inoculated with CTV. For propagation, selected buds from transgenic and non-transgenic control plants were grafted onto C. aurantium and C. limonia rootstock plants. CTV inoculation was performed via viruliferous aphids (Toxoptera citricida), and viral detection post-inoculation was performed through DASI-ELISA or RT-qPCR. After four inoculations, none of the transgenic lines tested showed complete resistance. However, viral multiplication was undetectable in some of the propagated clones. These resistant clones mainly came from transgenic ‘Valencia’ sweet orange plants grafted onto C. limonia rootstock containing the pCTV-CS gene construct. Although the tested viral inoculation method represents natural field infection conditions, the results did not differ significantly from those previously reported when the same transgenic lines were bud-graft inoculated. This finding indicates that the difficulties in producing CTV-resistant transgenic citrus lines may be unrelated to the inoculation method. Transgene expression level was quantified by RT-qPCR analysis and it was not possible to relate transgene mRNA level with resistance to the pathogen.     

Functional Characterization of Citrus Polygalacturonase-inhibiting Protein

A cDNA encoding a polygalacturonase-inhibiting protein gene (SaiPGIPA) was identified from the citrus cultivar Sainumphung (Citrus sp.), one of the most popular cultivars in northern Thailand. SaiPGIPA was expressed in Escherichia coli cells, and the functional properties of citrus PGIP were analyzed. The PGIP fusion protein inhibited by a maximum of about 60% of the endopolygalacturonase activity, and a mixture of the PGIP and fungal endopoly-galacturonase released oligogalacturonides from polygalacturonic acid. The mixture containing the oligogalactur-onides, endopolygalacturonase and PGIP induced expression of the PGIP gene and a chalcone synthase gene in citrus leaves. The mixture also induced resistance in cucumber leaves against Colletotrichum lagenarium. Received 5 September 2001/ Accepted in revised form 20 November 2001     

Enhanced resistance to citrus canker in transgenic sweet orange expressing the sarcotoxin IA gene

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subp. Citri (Xcc), is a serious disease reported in most citrus-producing areas around the world. Although different levels of field resistance to citrus canker have been reported in sweet oranges, they are usually not sufficient to provide adequate control of the disease. Ectopic over-expression of antibacterial genes is one of the potential strategies to increase plant resistance to bacterial diseases. Previous in vitro results showed that sarcotoxin IA, an antimicrobial peptide isolated from the flesh fly (Sarcophaga peregrina), can be efficient to control different plant pathogenic bacteria, including Xcc. Transgenic “Pera” sweet orange (Citrus sinensis [L.] Osbeck) plants constitutively expressing the sarcotoxin IA peptide fused to the PR1a signal peptide from Nicotiana tabacum for secretion in the intercellular space were obtained by Agrobacterium-mediated transformation using thin sections of mature explants. Citrus canker resistance evaluation in leaves of transgenic and non-transgenic plants was performed through inoculations with Xcc by infiltration and spraying. The Xcc population was up to 2 log unit lower in leaves of the transgenic plants compared to those of non-transgenic controls. Incidence of canker lesions was significantly higher in non-transformed controls (>10 lesions/cm²) than in the transgenic plants (<5 lesions/cm²) after injection infiltration or spraying with Xcc inoculum. Accumulation of sarcotoxin IA peptide in sweet orange tissue did not cause any deleterious effects on the growth and development of the transgenic plants, indicating this approach is suitable to provide resistance to citrus canker.     

A combined indexing and budwood propagation method for citrus

Several budwood-transmitted citrus diseases, including citrus tristeza virus, citrus psorosis, citrus impietratura and a range of citrus viroids, were tested both visually and biochemically on a combined indicator (CInd) plant consisting of an Alemow (Citrus macrophylla) rootstock grafted with Etrog citron (C.medica) and Sour orange (C.aurantium) or Sweet orange (C.sinensis) buds. Indexing on CInd plants is economical for limited testing space; an additional advantage is that, by collecting budwood directly from the CInd plants, the risk of diagnostic failure due to uneven pathogen distribution in the budwood source tree is considerably reduced.     

Molecular characterization of Citrus yellow mosaic badnavirus (CMBV) isolates revealed the presence of two distinct strains infecting citrus in India

Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the etiologic agent of yellow mosaic disease of citrus in India. The disease was initially reported from the southern parts of India and has now spread to other parts of the country. It is a serious disease of sweet orange (Citrus sinensis) in southern India, where it causes significant yield losses. During a recent survey of citrus groves in the Nagpur region, central India, characteristic mosaic symptoms were observed in mandarin orange (Citrus reticulata) and sweet orange. Virus transmission studies, electron microscopy, PCR amplification and sequencing of cloned PCR products from samples showing mosaic symptoms confirmed the presence of a badnavirus. The CMBV–Nagpur isolate could be transmitted to the Rangpur lime (C. limonia) and acid lime (Citrus aurantifolia) by graft inoculation. Sequence analysis of a segment of ORF-III region and intergenic region (IR) of the viral genome revealed that CMBV–Nagpur isolate formed a distinct clade along with some previously reported isolates that are known to infect acid lime and Rangpur lime. CMBV isolates that infect citrus species other than the acid lime and Rangpur lime formed a second clade. Based on the transmission studies and phylogenetic analyses, it was concluded that at least two strains of CMBV exist in India currently.     

Evidence for resistance to Citrus tristeza virus in pomelo (Citrus maxima Merr.) grown in Darjeeling and Sikkim hills of India

Darjeeling and Sikkim hills of India are well known for production of mandarin orange (Citrus reticulata). The recent spread of Citrus tristeza virus (CTV) has threatened the citrus cultivation in this region. During a survey in Darjeeling and Sikkim hills, pomelo trees were recorded as CTV free. Since pomelo trees did not show any disease appearance, a study was undertaken to ascertain whether they are resistant to CTV infection or resistant to aphid feeding or both. Toxoptera citricida, the most efficient aphid vector and which is abundantly present in this region, did not feed on pomelo when other Citrus species such as mandarin, kagzi lime and rough lemon were available. Additionally, CTV isolates of Darjeeling and Sikkim hills were not transmissible to pomelo either by T. citricida or grafting. We report for the first time that pomelo is resistant to isolates of CTV present in this region.     

Copper resistance genes from different xanthomonads and citrus epiphytic bacteria confer resistance to Xanthomonas citri subsp. citri

Genetic exchange is considered to be an important process in the selective adaptation of microorganisms to shifting and challenging environmental conditions. As a consequence of the copious use of copper bactericides, many species of plant pathogenic bacteria, including Xanthomonas citri subsp. citri (Xcc), have developed resistance to copper. This study assesses whether copper resistant (Cu^R) strains of other Xanthomonas species and citrus epiphytic bacteria pose a risk for the development of copper resistance in Xcc. Cu^R epiphytic bacteria were isolated on MGY agar from citrus leaves collected in two citrus groves treated with copper bactericides in Florida. Horizontal gene transfer of copper resistance genes was investigated within different Xanthomonas species and from citrus epiphytic bacteria to Xanthomonas. Cu^R epiphytic bacteria from citrus were screened for the presence of copper resistance genes homologous to copL, copA and copB genes from Xcc and characterized regarding tolerance to copper. Copper resistance determinants from a citrus epiphytic strain of Stenotrophomonas maltophilia (Stm) were cloned and expressed in Xcc and other Xanthomonas strains. Copper resistance genes in Xcc were determined to be present on a large (~300?kb) conjugative plasmid. Cu resistance was transferred via conjugation from two copper resistant citrus strains, Xcc and X. alfalfae subsp. citrumelonis (Xac), and two tomato pathogens, X. euvesicatoria (Xe) and X. perforans (Xp), to Xcc. PCR analysis revealed that two Cu^R strains from citrus, an epiphytic Xanthomonas ssp. and a strain of Stm, harboured homologs of the copper resistance genes found in Cu^R Xcc. The introduction of copLAB gene cluster from Stm into different xanthomonads conferred copper resistance to sensitive strains of Xcc, Xac, Xe and Xp. Based on these results there is a low, but significant, likelihood of horizontal gene transfer of copper resistance genes from other xanthomonads or epiphytic bacteria to Xcc in nature.