H. R. Azad

Characterization of Regulatory Pathways in Xylella fastidiosa: Genes and Phenotypes Controlled by algU

ABSTRACT Many virulence genes in plant bacterial pathogens are coordinately regulated by “global” regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierces disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.

Bacterial blight of broccoli raab : A new disease caused by a pathovar of Pseudomonas syringae

Bacterial blight is a new disease of broccoli raab or rappini (Brassica rapa subsp. rapa) that has developed on commercially grown crops in the Salinas Valley (Monterey County) in California. Symptoms consist of small, angular, water-soaked flecks on lower foliage that are visible from both adaxial and abaxial sides of the leaves. These flecks expand and become surrounded by bright yellow borders. With time, multiple leaf spots coalesce and result in large, irregular necrotic areas, extensive leaf yellowing, and eventual leaf death. If symptoms develop on the uppermost leaves attached to the inflorescence, the shoot loses market quality and will not be harvested. Pseudomonas syringae was consistently isolated from symptomatic plants, and selected strains caused similar symptoms when inoculated onto broccoli raab test plants. Broccoli raab strains caused leaf spot symptoms on nine other Cruciferous plants, as well as on three grass species (California brome, oat, and common timothy). Conversely, broccoli raab was not infected by P. syringae pathovars coronafaciens, maculicola, and tomato. Broccoli raab strains were positive for coronatine toxin production. Fatty acid analyses indicated that the P. syringae from broccoli raab was most closely related to P. syringae pvs. coronafaciens and maculicola, but its distinct host range suggests that it may be considered a separate pathovar.

First Report of Bacterial Leaf Spot of Spinach Caused by a Pseudomonas syringae Pathovar in California

In 2000 and 2001, a new disease was observed on commercial spinach (Spinacia oleracea) in the Salinas Valley, Monterey County, CA. Initial symptoms were water-soaked, irregularly shaped leaf spots (2 to 3 mm diameter). As the disease developed, spots enlarged to as much as 1 to 2 cm, were vein-delimited, and turned dark brown. Faint chlorotic halos sometimes surrounded the spots. Death of large areas of the leaf occurred if spots coalesced. Spots were visible from the adaxial and abaxial sides of leaves, and no fungal structures were observed. The disease occurred on newly expanded and mature foliage. No fungi were isolated from the spots. However, cream-colored bacterial colonies were consistently isolated on sucrose peptone agar, and these strains were nonfluorescent on Kings medium B. Strains were positive for levan and negative for oxidase, arginine dihydrolase, and nitrate reductase. Strains did not grow at 36°C, did not rot potato slices, but induced a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Turk). These results suggested the bacterium was similar to Pseudomonas syringae. Fatty acid methyl ester (FAME) analysis (MIS-TSBA 4.10, MIDI Inc., Newark, DE) indicated the strains were highly similar (80.1 to 89.3%) to P. syringae pv. maculicola. However, in contrast to P. syringae pv. maculicola, the spinach strains did not utilize the carbon sources erythritol, L+tartrate, L lactate, and DL-homoserine. Pathogenicity of 10 strains was tested by growing inoculum in nutrient broth shake cultures for 48 h, diluting to 106 CFU/ml, and spraying 4-week-old plants of spinach cv. Bossanova. Control plants were sprayed with sterile nutrient broth. After 5 to 8 days in a greenhouse (24 to 26°C), leaf spots identical to those observed in the field developed on cotyledons and true leaves of inoculated plants. Strains were reisolated from the spots and identified as P. syringae. Control plants remained symptomless. The 10 strains were also inoculated on beet (Beta vulgaris), Swiss chard (Beta vulgaris subsp. cicla), cilantro (Coriandrum sativum), and spinach. Spinach showed leaf spots after 8 days; however, none of the other plants developed symptoms. Two strains were inoculated onto spinach cvs. Califlay, Lion, Nordic IV, Polka, Resistoflay, Rushmore, RZ 11, Spinnaker, Springfield, Viroflay, and Whitney. Leaf spot developed on all cultivars, and the pathogen was reisolated. Because the FAME data indicated a similarity between the spinach pathogen and P. syringae pv. maculicola, we inoculated sets of spinach cv. Bolero, cabbage (Brassica oleracea subsp. capitata cv. Grenedere), and cauliflower (Brassica oleracea subsp. botrytis cv. White Rock) with three P. syringae pv. maculicola and three spinach strains. Cabbage and cauliflower developed leaf spots only when inoculated with P. syringae pv. maculicola; spinach had leaf spots only when inoculated with the spinach strains. All inoculation experiments were done twice, and the results of the two tests were the same. To our knowledge, this is the first report of bacterial leaf spot of spinach in California caused by a nonfluorescent P. syringae, and the first record of this disease in the United States. Biochemical characteristics and limited host range of the pathogen indicate the California strains are likely the same as the P. syringae pv. spinaciae pathogen that was reported in Italy (1) and Japan (2). References: (1) C. Bazzi et al. Phytopathol. Mediterr. 27:103, 1988. (2) K. Ozaki et al. Ann. Phytopathol. Soc. Jpn. 64:264, 1998.

Xanthomonas Leaf Spot of Catnip: A New Disease Caused by a Pathovar of Xanthomonas campestris

Xanthomonas leaf spot is a new disease that has occurred on catnip (Nepeta cataria). Catnip is grown commercially in California for use as herbs, seasonings, and tea. This disease has developed recently on catnip transplants that are produced in enclosed greenhouses. Symptoms consist of small brown flecks that are visible from both sides of a leaf. The flecks later develop into larger, dark brown, angular leaf spots. Severe infection reduced the quality and marketability of the transplants. Xanthomonas campestris, as identified by biochemical, physiological, and molecular tests, was consistently isolated from symptomatic plants, and selected strains caused similar symptoms when inoculated onto catnip test plants. However, catnip strains failed to cause any symptoms when inoculated onto nine other plants in the Lamiaceae family and five other hosts of known X. campestris pathovars. Catnip plants showed no symptoms when inoculated with X. campestris pvs. campestris, carotae, and vesicatoria. Catnip also was not susceptible to the X. campestris pathogen isolated from lavender. This is the first report of a bacterial disease of catnip caused by a Xanthomonas pathogen, and the catnip strains may be a new and distinct pathovar of X. campestris.

Diseases, pests, and abiotic disorders of greenhouse-grown water spinach (Ipomoea aquatica) in Ontario and California

Water spinach, a specialty vegetable and a member of the sweet potato family, is cultivated for Asian markets in California (United States) and Ontario (Canada). Foliar diseases of this vegetable observed in commercial greenhouses of Ontario in 1993 and 1994, as well as in greenhouses of California in 1998, were attributed respectively to Phyllosticta ipomoeae, Cercospora ipomoeae, and Pseudomonas syringae pv. syringae in the present study. In Ontario, C. ipomoeae infected less than 1% of plants, with less than 25% of the foliage affected, while Phyllosticta ipomoeae occurred on 100% of plants in some greenhouses, with leaf spotting affecting up to 25% of the foliage. In California, Pseudomonas syringae pv. syringae occurred on less than 25% of plants with up to 25% of the foliage affected. In Ontario, no virus was detected in eight suspect foliage samples while damage from vectors of plant viruses such as the green peach aphid (Myzus persicae) was minor and from Western flower thrips (Frankliniella occidentalis) was moderate. Edema affected water spinach at four sites in Ontario in 1994, with both the incidence and severity exceeding 75% at one site. For fungal and bacterial pathogens, Kochs postulates were fulfilled by inoculation and reisolation. Morphological characteristics of isolates in vitro and in vivo were used to identify the fungal pathogens, and LOPAT tests and fatty acid analyses were applied to identify the bacterial pathogen. Symptoms and etiology are discussed. This is the first report of these diseases, pests, and abiotic disorders on water spinach in Ontario and California.

A new ranunculus disease caused by Xanthomonas campestris

A new disease of ranunculus (Ranunculus asiaticus) was observed on several cultivars in commercial fields in San Diego and Riverside Counties, California. Symptoms included pin-point to large irregular necrotic lesions on leaves and stems and occasionally black patches along the internal margins of leaflets in association with vein chlorosis. Xanthomonas campestris was consistently isolated from diseased tissues. X. campestris was also isolated from tubers and seeds of naturally infected plants, which suggests a means by which the pathogen is spread in the industry. One-year-old tubers of two cultivars (Picotee and Rose) were contaminated at frequencies of 4 and 7%, respectively. The frequency of seed contamination for 11 cultivars ranged from 1.1 to 16%. Symptoms appeared on inoculated ranunculus plants as early as 3 and as late as 22 days after inoculation, depending on the method of inoculation, temperature, and available moisture. Recovery of the bacterium from the tubers of plants inoculated and kept under different moisture and temperature conditions was 6.6 to 13.3%. Amplification of a DNA fragment specific for hrp genes by polymerase chain reaction for each strain and further analysis of the amplification product by restriction endonuclease digestion suggested that the ranunculus strains were closely related to each other and to X. c. pv. campestris ; however, pathogenicity tests indicated that the ranunculus strains could be a different pathovar.