Biological control of bacterial spot of tomato under field conditions at several locations in North America

Following the relatively successful biological control of bacterial speck of tomato under field conditions at several locations (Phytopathology 92 (2002) 1284), similar selection and testing strategies were employed in an effort to isolate an effective biological control agent for bacterial spot of tomato. Fifty potential biological control agents were isolated from tomato foliage in Alabama (AL) and Florida (FL) and tested under greenhouse conditions in AL for the ability to reduce the foliar severity of bacterial spot of tomato (Lycopersicon esculentum), which is caused by either Xanthomonas campestris pv. vesicatoria or Xanthomonas vesicatoria. Three pseudomonads that provided protection against bacterial speck also were included in the tests. The strains which were most efficacious (i.e., high mean percentage reduction) and consistent (i.e., low standard deviation) in reducing bacterial spot severity in repeated greenhouse experiments were selected for field experiments conducted over the period 1996–1998. Among these strains were Cellulomonas turbata BT1, which provided the highest mean reduction in disease severity [45.2% (SD = 21.0)], and Pseudomonas syringae Cit7 [36.4% (SD = 12.2)], which was the most consistent. Field experiments were conducted in Shorter, AL; Bradenton and Sanford, FL; Clinton, North Carolina; Wooster, Ohio; and London, Ontario, Canada. The highest mean reductions in severity of bacterial spot on foliage, averaged across all locations, were provided by P. syringae Cit7 [28.9% (SD = 11.6)] and Pseudomonas putida B56 [23.1% (SD = 7.4)]. The efficacy and consistency of P. syringae Cit7 against bacterial spot were very similar to those achieved against bacterial speck [28.3% (SD = 12.7)] (Phytopathology 92 (2002) 1284). Unfortunately, neither the bacterial strains nor the standard copper bactericides consistently reduced disease incidence on fruit.     

Expression ofMbR4, a TIR-NBS type of appleR Gene, confers resistance to bacterial spot disease inArabidopsis

A single disease resistance gene candidate,MbR4, was isolated from the wild-type apple speciesMalus baccta. This gene was predicted to encode motifs characteristic of the Toll Interleukin 1 Receptor (TIR) — Nucleotide Binding Site (NBS) of theR gene. Starting with an isolated cDNA clone, genomic clones were obtained via inverse polymerase chain reaction (IPCR). TheMbR4 gene has a single open reading frame (ORF) of 2178 nucleotides, a 41-b untranslated 5’ region, a 21-b untranslated 3’ region, and a predicted protein of 726 amino acids (82 kDa). Its deduced amino acid sequence resembles the N protein of tobacco and the NL25 protein of potato. Ectopic expression ofMbR4 induced enhanced resistance in transgenicArabidopsis plants against the virulent pathogen,Pseudomonas syringae pv.tomato DC3000. Microarray analysis confirmed the induction of defense-related gene expression in pathogen-free 35S::MbR4 heterologousArabidopsis plants, thereby indicating that theMbR4 gene likely activates a pathogen-independent resistance pathway, rather than a gene-for-gene pathway. Our results suggest thatMbR4 plays a role in theR gene, and may be a source of resistance for cultivated apple species.     

Identification of QTL associated with resistance to bacterial spot race T4 in tomato

Bacterial spot of tomato (Solanum lycopersicum L.), caused by several Xanthomonas sp., is a serious but difficult disease to control by chemical means. Development of resistance has been hindered by emergence of races virulent to tomato, by the quantitative inheritance of resistance, and by a low correlation between seedling assays and resistance in the field. Resistance to multiple races, including race T4, has been described in the S. lycopersicum var. cerasiformae accession PI 114490. We used molecular markers to identify associations with quantitative trait loci (QTL) in an elite inbred backcross (IBC) population derived from OH 9242, PI 114490 and Fla. 7600, a breeding line with tomato accession Hawaii 7998 (H7998) in its pedigree. Race T4 resistance has also been described in the advanced breeding lines Fla. 8233, Fla. 8517, and Fla. 8326, and a selective genotyping approach was used to identify introgressions associated with resistance in segregating progeny derived from crosses with these lines. In the IBC population, loci on chromosomes 11 and 3, respectively, explained as much as 29.4 and 4.8% of resistance variation. Both these loci were also confirmed by selective genotyping: PI 114490 and H7998 alleles on chromosome 11 each provided resistance. The PI 114490 allele on chromosome 3 was confirmed in the Fla. 8517 population, and an allele of undetermined descent was confirmed at this locus in the Fla. 8326 population. A chromosome 12 allele was associated with susceptibility in the Fla. 8517 population. Additional loci contributing minor effects were also implicated in the IBC population or by selective genotyping. Selection for the major QTL in a marker-directed phenotyping approach should significantly improve the efficiency of breeding for resistance to bacterial spot race T4, although as yet undetected QTL would be necessary to carry out strict marker assisted selection.     

Molecular mechanisms involved in bacterial speck disease resistance of tomato

An important recent advance in the field of plant-microbe interactions has been the cloning of genes that confer resistance to specific viruses, bacteria, fungi or nematodes. Disease resistance (R) genes encode proteins with predicted structural motifs consistent with them having roles in signal recognition and transduction. The future challenge is to understand how R gene products specifically perceive defence-eliciting signals from the pathogen and transduce those signals to pathways that lead to the activation of plant defence responses. In tomatoes, the Pto kinase (product of the Pto R gene) confers resistance to strains of the bacterial speck pathogen, Pseudomonas syringae pv. tomato, that carry the corresponding avirulence gene avrPto. Resistance to bacterial speck disease is initiated by a mechanism involving the physical interaction of the Pto kinase and the AvrPto protein. This recognition event initiates signalling events that lead to defence responses including an oxidative burst, the hypersensitive response and expression of pathogenesis-related genes. Pto-interacting (Pti) proteins have been identified that appear to act downstream of the Pto kinase and our current studies are directed at elucidating the roles of these components.     

Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt disease

Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, is one of the most important diseases of banana (Musa sp.) and currently considered as the biggest threat to banana production in Great Lakes region of East and Central Africa. The pathogen is highly contagious and its spread has endangered the livelihood of millions of farmers who rely on banana for food and income. The development of disease resistant banana cultivars remains a high priority since farmers are reluctant to employ labor-intensive disease control measures and there is no host plant resistance among banana cultivars. In this study, we demonstrate that BXW can be efficiently controlled using transgenic technology. Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of banana. These transgenic lines were characterized by molecular analysis. After challenge with X. campestris pv. musacearum transgenic lines showed high resistance. About 67% of transgenic lines evaluated were completely resistant to BXW. These transgenic lines did not show any disease symptoms after artificial inoculation of in vitro plants under laboratory conditions as well as potted plants in the screen-house, whereas non-transgenic control plants showed severe symptoms resulting in complete wilting. This study confirms that expression of the Pflp gene in banana results in enhanced resistance to BXW. This transgenic technology can provide a timely solution to the BXW pandemic.     

Integrated biological control of bacterial speck and spot of tomato under field conditions using foliar biological control agents and plant growth-promoting rhizobacteria

Integration of foliar bacterial biological control agents and plant growth promoting rhizobacteria (PGPR) was investigated to determine whether biological control of bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, and bacterial spot of tomato, caused by Xanthomonas campestris pv. vesicatoria and Xanthomonas vesicatoria, could be improved. Three foliar biological control agents and two selected PGPR strains were employed in pairwise combinations. The foliar biological control agents had previously demonstrated moderate control of bacterial speck or bacterial spot when applied as foliar sprays. The PGPR strains were selected in this study based on their capacity to induce resistance against bacterial speck when applied as seed and soil treatments in the greenhouse. Field trials were conducted in Alabama, Florida, and California for evaluation of the efficacy in control of bacterial speck and in Alabama and Florida for control of bacterial spot. The foliar biological control agent P. syringae strain Cit7 was the most effective of the three foliar biological control agents, providing significant suppression of bacterial speck in all field trials and bacterial spot in two out of three field trials. When applied as a seed treatment and soil drench, PGPR strain Pseudomonas fluorescens 89B-61 significantly reduced foliar severity of bacterial speck in the field trial in California and in three of six disease ratings in the field trials in Alabama. PGPR strains 89B-61 and Bacillus pumilus SE34 both provided significant suppression of bacterial spot in the two field trials conducted in Alabama. Combined use of foliar biological control agent Cit7 and PGPR strain 89B-61 provided significant control of bacterial speck and spot of tomato in each trial. In one field trial, control was enhanced significantly with combined biological control agents compared to single agent inoculations. These results suggest that some PGPR strains may induce plant resistance under field conditions, providing effective suppression of bacterial speck and spot of tomato, and that there may be some benefit to the integration of rhizosphere-applied PGPR and foliar-applied biological control agents.     

A centenary for bacterial spot of tomato and pepper

Disease symptomsSymptoms include water‐soaked areas surrounded by chlorosis turning into necrotic spots on all aerial parts of plants. On tomato fruits, small, water‐soaked, or slightly raised pale‐green spots with greenish‐white halos are formed, ultimately becoming dark brown and slightly sunken with a scabby or wart‐like surface.Host rangeMain and economically important hosts include different types of tomatoes and peppers. Alternative solanaceous and nonsolanaceous hosts include Datura spp., Hyoscyamus spp., Lycium spp., Nicotiana rustica, Physalis spp., Solanum spp., Amaranthus lividus, Emilia fosbergii, Euphorbia heterophylla, Nicandra physaloides, Physalis pubescens, Sida glomerata, and Solanum americanum.Taxonomic status of the pathogenDomain, Bacteria; phylum, Proteobacteria; class, Gammaproteobacteria; order, Xanthomonadales; family, Xanthomonadaceae; genus, Xanthomonas; species, X. euvesicatoria, X. hortorum, X. vesicatoria.Synonyms (nonpreferred scientific names) Bacterium exitiosum, Bacterium vesicatorium, Phytomonas exitiosa, Phytomonas vesicatoria, Pseudomonas exitiosa, Pseudomonas gardneri, Pseudomonas vesicatoria, Xanthomonas axonopodis pv. vesicatoria, Xanthomonas campestris pv. vesicatoria, Xanthomonas cynarae pv. gardneri, Xanthomonas gardneri, Xanthomonas perforans.Microbiological propertiesColonies are gram‐negative, oxidase‐negative, and catalase‐positive and have oxidative metabolism. Pale‐yellow domed circular colonies of 1–2 mm in diameter grow on general culture media.DistributionThe bacteria are widespread in Africa, Brazil, Canada and the USA, Australia, eastern Europe, and south‐east Asia. Occurrence in western Europe is restricted.Phytosanitary categorizationA2 no. 157, EU Annex designation II/A2.EPPO codesXANTEU, XANTGA, XANTPF, XANTVE.     

Identification and fine-mapping of a bacterial brown spot disease resistance gene in maize

Bacterial brown spot (BBS) in maize (Zea mays L.) is caused by Pseudomonas syringae pv. syringae Van Holl (Pss). In China, this disease is not prevalent in maize at present. Here, we report the identification and fine mapping of the gene, referred to as Psy1, which confers resistance to BBS. An F₂ population, derived from the cross P25/F349, was used for linkage analysis and mapping of the resistance gene Psy1. Analysis of a BC₈F₂ population, derived from the same parents, confirmed that Psy1 was located on chromosome 10L and inherited as a single dominant gene. For fine mapping of Psy1, two introgression lines, X41 and X44, homozygous at the resistant gene locus, were introduced to hybridize with the susceptible parent P25 respectively, and developed a mixed BC₁ population. We found the closest markers to Psy1 are EST1 and FG29-3, which located on two adjacent BACs respectively, based on the B73 BAC sequence. Sequence analysis of these two BAC sequences (~300 kb) revealed the presence of a homologous sequence of receptor-like kinase. Also a co-segregation marker was developed based on this homologous sequence. These results will be useful for cloning of Psy1 and for transferring or pyramiding Psy1 through MAS in maize breeding programs.     

Characterization of IS476 and its role in bacterial spot disease of tomato and pepper.

IS476 is an endogenous insertion sequence present in copper-tolerant strains of Xanthomonas campestris pv. vesicatoria. Sequence analysis has revealed that the element is 1,225 base pairs in length, has 26-base-pair inverted repeats, and causes a 4-base-pair target site duplication upon insertion into the avirulence gene avrBs1. Comparison of the full-length sequence with sequences in the National Biomedical Research Foundation and National Institutes of Health data bases showed that one of the predicted IS476 proteins is partially homologous to the putative transposase of IS3 from Escherichia coli, and the inverted repeats of IS476 have significant homology to the inverted repeats of the IS51 insertion sequence of Pseudomonas syringae pv. savastanoi. A transposition assay based on the insertional inactivation of the sacRB locus of Bacillus subtilis was used to demonstrate that one of the three copies of IS476 residing on the 200-kilobase copper plasmid pXVCU1 is capable of transposition in several strains of Xanthomonas campestris. The position of IS476 insertion in several avrBs1 mutants was established and was shown to influence both induction of hypersensitivity and bacterial growth in planta.     

A spreading colony formed method for rapid evaluation of dicarboximide fungicides resistance level of field tobacco brown spot disease

A new method (spreading colony formed method) for rapidly identifying and evaluating the dicarboximide fungicides resistance level of field tobacco spot brown disease caused byAlternaria longipes was developed. Two typical colonies with distinct differences in colony morphology on media containing 5 μg/ml dicarboximide fungicides dimethachlon (CAS registration number: 24096-53-5) were discovered by using this method. The two typical colonies were named spreading colony and dense pad colony, respectively. Isolates (250) ofA. longipes were quickly separated by this method, and their growth properties (including the sensitivity to dimethachlon, the cross-resistance to phenylpyrroles fludioxonil and hyphal development) were examined. Our results indicated that (1) monospore isolates from spreading colonies and dense pad colonies were respectively resistant and sensitive to dimethachlon; (2) resistant and sensitive isolates formed respectively spreading colonies and dense pad colonies on dimethachlon media. Furthermore, molecular experiments confirmed the spreading colony formed method reliable. In conclusion, field resistant isolates and resistant situation in population level of field tobacco spot brown disease could be exactly and timely determined and evaluated by spreading colony formed method.     

Biological control of bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis

Xanthomonas campestris pv. vesicatoria strain 2 was isolated from infected tomato seedlings grown in open field in Egypt. This strain produced irregular yellow-necrotic areas on tomato leaves and spotting of the stem. In an attempt to control this disease biologically, four experiments were conducted and tomato seedlings were pretreated, before the pathogen, with either of two antagonistic strains of Rahnella aquatilis through leaves, roots, soil or seeds. In all experiments, seedlings pretreated with R. aquatilis showed reduced susceptibility toward X. c. pv. vesicatoria. They also contained reduced protein concentration and showed reduced number of protein bands in SDS-PAGE analysis as well as increased fresh and dry weight relative to control seedlings inoculated with the pathogen only. This indicates that R. aquatilis reduced the deleterious effect and the stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R. aquatilis was the most effective method in disease reduction which could be attributed to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts of fresh and dry weight ere obtained from seed treatment, which might suggest that bacterial seed inoculation provides earlier protection than could be achieved with foliar, soil or root treatment.     

Enhancement of the citrus immune system provides effective resistance against Alternaria brown spot disease

In addition to basal defense mechanisms, plants are able to develop enhanced defense mechanisms such as induced resistance (IR) upon appropriate stimulation. We recently described the means by which several carboxylic acids protect Arabidopsis and tomato plants against fungi. In this work, we demonstrate the effectiveness of hexanoic acid (Hx) in the control of Alternaria brown spot (ABS) disease via enhancement of the immune system of Fortune mandarin.     

Effect of acidic electrolyzed water on the viability of bacterial and fungal plant pathogens and on bacterial spot disease of tomato

Acidic electrolyzed water (AEW), known to have germicidal activity, was obtained after electrolysis of 0.045% aqueous solution of sodium chloride. Freshly prepared AEW (pH 2.3-2.6, oxidation-reduction potential 1007-1025 mV, and free active chlorine concentration 27-35 ppm) was tested in vitro and (or) on tomato foliage and seed surfaces for its effects on the viability of plant pathogen propagules that could be potential seed contaminants. Foliar sprays of AEW were tested against bacterial spot disease of tomato under greenhouse and field conditions. The viability of propagules of Xanthomonas campestris pv. vesicatoria (bacterial spot pathogen), Streptomyces scabies (potato scab pathogen), and Fusarium oxysporum f.sp. lycopersici (root rot pathogen) was significantly reduced 4-8 log units within 2 min of exposure to AEW. Immersion of tomato seed from infected fruit in AEW for 1 and 3 min significantly reduced the populations of X. campestris pv. vesicatoria from the surface of the seed without affecting seed germination. Foliar sprays of AEW reduced X. campestris pv. vesicatoria populations and leaf spot severity on tomato foliage in the greenhouse. In the field, multiple sprays of AEW consistently reduced bacterial spot severity on tomato foliage. Disease incidence and severity was also reduced on fruit, but only in 2003. Fruit yield was either enhanced or not affected by the AEW sprays. These results indicate a potential use of AEW as a seed surface disinfectant or contact bactericide.     

A CCCH-type zinc finger nucleic acid-binding protein quantitatively confers resistance against rice bacterial blight disease

Bacterial blight is a devastating disease of rice (Oryza sativa) caused by Xanthomonas oryzae pv oryzae (Xoo). Zinc finger proteins harboring the motif with three conserved cysteine residues and one histidine residue (CCCH) belong to a large family. Although at least 67 CCCH-type zinc finger protein genes have been identified in the rice genome, their functions are poorly understood. Here, we report that one of the rice CCCH-type zinc finger proteins, C3H12, containing five typical CX(8)-CX(5)-CX(3)-H zinc finger motifs, is involved in the rice-Xoo interaction. Activation of C3H12 partially enhanced resistance to Xoo, accompanied by the accumulation of jasmonic acid (JA) and induced expression of JA signaling genes in rice. In contrast, knockout or suppression of C3H12 resulted in partially increased susceptibility to Xoo, accompanied by decreased levels of JA and expression of JA signaling genes in rice. C3H12 colocalized with a minor disease resistance quantitative trait locus to Xoo, and the enhanced resistance of randomly chosen plants in the quantitative trait locus mapping population correlated with an increased expression level of C3H12. The C3H12 protein localized in the nucleus and possessed nucleic acid-binding activity in vitro. These results suggest that C3H12, as a nucleic acid-binding protein, positively and quantitatively regulates rice resistance to Xoo and that its function is likely associated with the JA-dependent pathway.     

Loss of SLP-76 expression within myeloid cells confers resistance to neutrophil-mediated tissue damage while maintaining effective bacterial killing

The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is an adaptor molecule critical for immunoreceptor and integrin signaling in multiple hemopoietic lineages. We showed previously that SLP-76 is required for neutrophil function in vitro, including integrin-induced adhesion and production of reactive oxygen intermediates, and to a lesser extent, FcgammaR-induced calcium flux and reactive oxygen intermediate production. It has been difficult to determine whether SLP-76 regulates neutrophil responses in vivo, because Slp-76(-/-) mice exhibit marked defects in thymocyte and vascular development, as well as platelet and mast cell function. To circumvent these issues, we generated mice with targeted loss of SLP-76 expression within myeloid cells. Neutrophils obtained from these animals failed to respond to integrin activation in vitro, similar to Slp-76(-/-) cells. Despite these abnormalities, SLP-76-deficient neutrophils migrated normally in vivo in response to Staphylococcus aureus infection and efficiently cleared micro-organisms. Interestingly, SLP-76-deficient neutrophils did not induce a robust inflammatory response in the localized Shwartzman reaction. Collectively, these data suggest that disruption of integrin signaling via loss of SLP-76 expression differentially impairs neutrophil functions in vivo, with preservation of migration and killing of S. aureus but reduction in LPS-induced tissue damage and vascular injury.     

Pn-AMPs,the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato,Lycopersicum esculentum

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.