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Gram-negative plant pathogenic bacteria are secreting into plant cell a special type of pathogeni city-related proteins called effectors. They are capable of suppressing plant innate immunity or stimulating synthesis and export of metabolites desired by the pathogen. We identified a number of effector-coding genes typical of xanthomonads analyzing 8 completely sequenced genomes of genus Xanthomonas. Using representative collection provided by Russian Research Institute of Phytopathology we identified genetic diversity of effector gene loci in population of Xanthomonas bacteria. Patterns of effector genes were identified for individual strains and statistic linkage between particular genes and race of the pathogen was established. For the first time several untypical effector genes were found in strains of Xanthomonas campestris pv. campestris.

Our highly concentrated monoculture makes crops vulnerable to pests and diseases. An increase in emerging non-indigenous bacterial diseases poses a real threat to US agriculture. The United States has 100,000 miles of shoreline and 6,000 miles of border, making possible easy introduction of crop pests and diseases. Most threatening to crops are the cross-domain enteric bacteria. In contrast to animals, crops have hundreds of major diseases and development of molecular-based detection protocols for each pathogen is impossible with current technology. Rathayibacter toxicus, a neurotoxin-producing bacterium transmitted by a seed gall nematode, is an example of a high-risk Select Agent. The bacterium infects seeds of grasses without showing any symptoms, often resulting in the death of grazing cattle. A prerequisite for the control of any disease is sensitive detection and proper identification of the causal organism. Detecting bacteria in samples of plants showing symptoms is relatively simple, whereas detection in asymptomatic tissues is difficult due to the extremely low numbers of the target pathogen present. Rapid serological assays work well with symptomatic tissues but not from asymptomatic tissue when bacteria levels are below sensitivity limits. Classical agar-plating assays are 1,000 fold more sensitive then serology or PCR. However, agar plating assays take from 3 to 5 days and require pathogenicity tests to confirm the identity. PCR-based assays allow for rapid, accurate identification but are insensitive due to use of 1 microL sample in comparison to 100 microL used for agar plating. To overcome this disadvantage, an enrichment technique termed BIO-PCR can be used in combination with agar plating for detection with asymptomatic tissues. The key to developing a successful BIO-PCR protocol is to determine the time required for development of pin point-size colonies to appear. For most plant pathogens 15 to 24 hours is sufficient time, whereas for the cross-domain bacteria only 1 to 2 hours is needed. For greater sensitivity, BIO-PCR can be combined with 96-well microliter plates with membranes to detect a single viable cell per 10 mL of an aqueous sample.

The survival and half-life of Clavibacter michiganensis ssp. michiganensis (C. michiganensis), the causal agent of bacterial canker of tomato, were determined in infected plant debris under natural field conditions in California, Ohio and Morocco using a semiselective agar medium. The organism survived significantly longer in tomato stems left on the soil surface than in stems buried in the soil at all locations studied. The pathogen was recovered in high amounts from tomato stems left on the soil surface for 314 days in Ohio and California, USA, and for 194 and 132 days in Melk Zhar and Ait Melloul, Morocco, respectively; it was recovered from stems buried in the soil for up to 314 days in Ohio, up to 240 days in California, and up to 60 days in Ait Melloul and Melk Zhar. The half-life of the pathogen in stems left on the soil surface ranged from 23·2 to 24·8 days in the USA, and from 7·8 to 12·3 days in Morocco, whereas the half-life in buried stems ranged from 14·0 to 16·7 days in the USA and from 3·7 to 9·5 days in Morocco. Based on the half-life data, the predicted survival times of C. michiganensis in stems on the soil surface in Ohio, California, Melk Zhar and Ait Melloul would be up to 822, 770, 424 and 261 days, respectively, while the predicted survival times in stems buried in the soil would be 541, 497, 305 and 128 days, respectively. These results show that the survival and half-life of C. michiganensis in plant debris are relatively long and are influenced by both tissue exposure and geographic location.

Acidovorax avenae subsp. citrulli (Pseudomonas pseudoalcaligenes subsp. citrulli), the causal agent of a watermelon seedling blight and fruit blotch (WFB), has emerged as a serious seedborne pathogen of watermelon and melons world-wide. The disease was first reported in 1965 in seedlings of several USDA Plant Introduction (PI) lines in Georgia. The pathogen was described 13 years later in diseased seedlings from two of the same PI seed sources in which Web and Goth had originally identified the disease. The disease remained known only in seedlings at the introduction station until Wall and Santos isolated a non-fluorescent pseudomonad from rotting fruit in Guam in 1987 and concluded it was the same organism. WFB was reported in Florida in 1989 and a year later emerged as a major economic disease of watermelon in the Southeast and Midwest USA. Since then, WFB has been reported in Turkey, Brazil, Australia, Japan, Korea, Thailand, and China infecting cantaloupe, melon, honeydew, pumpkin, and citron under field conditions. Because the original seedling strains were reported as giving a negative tobacco hypersensitive reaction and failed to rot fruit like the field disease strains, some have suggested that the originally described organism was not the same organism as that causing WFB. DNA fingerprinting of a collection of 121 strains using pulse field gel electrophoresis revealed considerable genetic diversity. All strains typed into two groups; over 80% were in Group I and the remaining, including the original type strain, in Group II. Control of the disease is based primarily on use of pathogen-free seed. Although attempts have been made to develop a routine seed assay to detect the organism in seeds, no reliable method is available. Contaminated seeds remain a problem for the seed industry.

Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3-kb EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T, indica and not from a morphologically similar smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2,3-kb fragment from total DNA of 17 T. indica isolates from India, Pakistan, and Mexico, DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia foetida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected, The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds. Appl Environ Microbiol

A polymerase chain reaction (PCR) method, hereafter referred to as BIO-PCR, that combines biological and enzymatic amplification of PCR targets and simplified procedures for sample processing is described for the detection of Pseudomonas syringae pv. phaseolicola in bean seed extracts. Seeds are soaked overnight following standard protocols and aliquots of the extracts are plated onto a general agar medium. After 45-48 hr of incubation, the plates are washed with water to remove bacterial cells and aliquots of a pooled washing are subjected to two consecutive rounds of PCR, without prior DNA extraction, using «nested» pairs of primers designed to amplify a segment of the organism's tox (phaseolotoxin) gene region. Positive detection was reproducibly obtained at near-limit concentrations of the pathogen in the seed wash. Advantages of BIO-PCR over existing PCR techniques include the elimination of false positives resulting from the presence of dead cells that may be present in the seed, elimination of false negatives due to potential PCR inhibitors in seed extracts, increased sensitivity of detection, and no need for DNA extraction prior to amplification. Accordingly, BIO-PCR should prove useful for routine detection of other bacterial pathogens of quarantine importance. The primers detected some nontoxigenic strains of the pathogen, which evidently contain part of or the entire tox gene cluster

Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker, was eradicated from naturally infected tomato (Lycopersicon esculentum) seeds that were soaked in 0.6 M HCl for 5 hr, in 0.25 or 0.50% acidified cupric acetate (ACA) for 20 min, or in water at 52 C for 20 min or at 56 C for 30 min. Other individual treatments failed to eradicate the pathogen. Most treatments reduced the saprophytic bacterial flora on tomato seeds; however, only the HCl treatment and treatment with ACA (0.25%) at 52 C for 20 min eradicated saprophytes. Germination of treated seed varied with the germination test (.)

A semiselective medium, called XOS, was developed to isolate Xanthomonas oryzae pv. oryzae and X. o. oryzicola from rice seed. The medium contains sucrose, peptone, monosodium glutamate, calcium nitrate, potassium phosphate, iron EDTA, cycloheximide, cephalexin, kasugamycin, methyl violet 2B, and agar. Recovery of X. o. oryzae, X. o. oryzicola and nonpathogenic Xanthomonas-like bacteria on XOS basal medium ranged from 98 to 162% (mean 121%) of the recovery on nutrient glucose agar after incubation at 28 C for 4 days. Addition of the selective inhibitors decreased recovery of non-xanthomonas saprophytes in rice seed extracts by over 53%, compared to XOS basal medium. Several saprophytic bacteria common to some Chinese seed lots were not significantly reduced; however, the growth of such bacteria was decreased enough to allow detection of the slower growing xanthomonads. Recovery of different pathogenic strains of the rice xanthomonads varied significantly on XOS. Detection of Xanthomonas spp. from seed was dependent on partial maceration of seed and incubation of the resulting extract at 5 C for 2 h. Extracts from rice seed collected from asymptomatic plants in California and China and from plants with symptoms of bacterial leaf blight in China were plated on XOS, and colonies suggestive of Xanthomonas were isolated. Various identification assays indicate that these strains belong to the genus Xanthomonas; however, the strains were nonpathogenic when inoculated on a susceptible rice cultivar. XOS is quantitatively and qualitatively superior to other available media for isolation of Xanthomonas spp. from rice seed

The nomenspecies Pseudomonas avenae Manns 1909 and Pseudomonas alboprecipitans Rosen 1922, causal agents of leaf blight of oats and leaf blight of corn, have been compared with respect to their physiological and biological properties as described in the early literature. Except for a few minor physiological characters, most of the properties were identical to those determined herein with P. alboprecipitans isolates obtained in Georgia and Florida and with the “lecto” type strain (ATCC 19860). All isolates of P. alboprecipitans were closely related genetically as judged from deoxyribonucleic acid (DNA)-DNA reassociation experiments. On the other hand, DNA of P. alboprecipitans failed to reassociate with DNA of fluorescent Pseudomonas syringae 5D417 and Pseudomonas andropogonis PA146 isolated from sorghum or the nonfluorescent Pseudomonas atrofaciens PA13 isolated from wheat. P. alboprecipitans isolates also possessed DNA of relatively high guanine plus cytosine (G+C) content (72.2 mol%) as compared to the 57.8 mol% G+C of P. atrofaciens and P. andropogonis.

Eleven strains of Pseudomonas coronafaciens, four strains of P. coronafaciens pathovar zeae, six strains of P. coronafaciens subsp. atropurpurea, two strains of P. striafaciens, and one Pseudomonas sp. were compared by physiological, biochemical, serological, and pathological tests. All strains tested utilized sucrose, glucose, fructose, glycerol, and caprate as sole carbon sources, hydrolyzed Tween 80, reduced litmus milk, and grew at 0°C. All strains were arginine dihydrolase and oxidase negative and all strains failed to hydrolyze starch, reduce nitrate, or grow at 41°C. Cells of all strains were immunofluorescent positive when stained by the indirect immunofluorescence test with antiserum to cells of P. coronafaciens or P. coronafaciens subsp. atropurpurea. No differences among strains were detected in test tube agglutination tests. No significant differences were detected in the susceptibility of Avena sativa (oats), Secale cereale (rye), Triticum aestivum (wheat), Hordeum vulgare (barley), Bromus inermis (smooth bromegrass), B. japonicus (Japanese brome), B. secalinus (chess brome), B. tectorum (cheatgrass), Agropyron repens (quackgrass), Zea mays (maize), or Phleum pratense (timothy) to the different strains. All strains produced toxin in Wooley medium except P. striafaciens and strains of P. coronafaciens subsp. atropurpurea from Italian ryegrass. The present division of strains of P. coronafaciens based on minor differences in pathogenicity and symptomology is untenable.

SUMMARY A requisite for development of bacterial phloem canker caused by Erwinia rubrifaciens is the presence of the highly susceptible Hartley cultivar; when interplanted with Hartley, the Franquette and Payne cultivars are sometimes attacked by the disease. The recently developed cultivars, Gustine and Howe, developed active cankers when inoculated, but not as extensive as those in Hartley. The age of the plant part is important in the disease. One-year-old Hartley trees and twigs of the current season on mature trees did not develop active cankers. The complete disease syndrome occurs only on trunks and primary (scaffold) branches. Extension of the cankers was most rapid during the summer when the temperature was high. This was correlated with the effect of temperature on growth of the bacterium in culture. At midday in summer the temperature of the cambial area of a shaded trunk was as much as 10dF (5.6 dC) below ambient temperature but it was commonly a few degrees above ambient at night. Breaks in the thick phelloderm of the trunks and branches are necessary for penetration of the pathogen to the inner bark. Of the several types of breaks commonly occurring, those produced by mechanical harvesting equipment and by sap-sucking birds were found to be infection sites. Bacteria occur in large numbers in a slimy substance which exudes through cracks and accumulates on the bark of infected trees. They survived for at least 123 days in the exudate and were disseminated laterally as far as 20 ft (6.1 m) in wind-blown rain. Bacteria were also transmitted from tree to tree in exudate contaminating the pads of mechanical harvesting equipment.

Black chaff, caused by Xanthomonas campestris pv. translucens(hereafter referred to as X. translucens), has caused large yield reductions in irrigated wheat in southern Idaho recently. A seed assay using a semiselective agar medium (XTS agar) for detection of the pathogen was recently described (Phytopathology 75:260–263). Field studies were conducted in 1983 and 1984 to quantify the relationship between the level of X. translucens contamination (naturally infected and/or infested) in the seed with black chaff development in the field. In both years, different levels of contamination in wheat seed lots were achieved by mixing contaminated seed with 0, 90, and 99% foundation seed. The foundation seed had a trace level of X. translucens.

Plants respond in many ways to invasion by pathogenic organisms. The plant’s response is often so specific that presumptive diagnosis can be made by symptoms alone. Diseases caused by fungal pathogens are generally easy to diagnose because the pathogen produces characteristic fruiting bodies or spores visible with a hand lens or light microscope. In contrast, cells of bacteria are difficult to observe under a light microscope. However, in many cases masses of cells can easily be seen oozing from freshly cut tissue. Some bacteria ooze in such great quantities that a microscope is not needed. For example, with P. solanacearum infected stem tissue can be placed in water in a graduate cylinder to visually observe bacteria streaming from the tissues.

Classification of plant pathogenic bacteria has always been in controversy. Traditionally, classification of plant bacteria has been based upon a few biochemical characters and pathogenicity. In practice, pathogenicity became the most important character. Basic studies on the nutrition, biochemistry, physiology and structure were largely ignored. If a bacterium was isolated from a plant and shown to be pathogenic, it was given a species rank. This system of naming new pathogens and their classification was accepted and worked fairly well until the International Committee on Systematic Bacteriology (ICSB) established 1980 as the new starting date for the nomenclature of bacteria.

Acidified cupric acetate soaks were tested for eradication of Xanthomonas campestris from naturally infected crucifer seeds. The pathogen was eradicated from seeds by soaking in 0.5% cupric acetate dissolved in 0.005 N acetic acid for 20 min at 35, 40, 45, and 50°C but not 25°C. Moreover, normal bacterial flora of crucifer seeds and the seed-borne Phoma lingam and Alternaria spp. were reduced by 95, 92, and 81%, respectively, after the cupric acetate treatment at 40°C. The seed germination percentage was generally reduced, but the amount of reduction depended upon the treatment temperature and plant cultivar. At 50°C, less than 50% of the seed of all 12 cultivars tested germinated, whereas at 40°C more than 50% of the seeds of most cultivars germinated. Treating seeds in cupric acetate at 40°C should prove useful for eradicating X. campestris from seeds of breeding lines and stock seed used for hybrid seed production. Furthermore, a significant reduction in total bacterial flora and seed-borne fungi suggests the usefulness of the treatment for other microorganisms associated with other seeds or foodstuffs.

Distribution et severite de la nervation noire des Cruciferes en Thailande. Collecte de souches representatives en vue d'une etude serologique pour identification de Xanthomonas campestris

SCHAAD, N. W., D. R. SUMNER, and G. 0. WARE. 1980. Influence of temperature and light on severity of bacterial blight of corn, oats, and wheat. Plant Disease 64:481-483. Two experiments using five strains of Pseudomonas avenae and five day/night temperature regimens were conducted to determine the influence of temperature and light on the severity of bacterial leaf blight of corn, wheat, and oats. The first experiment showed that light had no effect on lesion development. In the second experiment, plant growth data for corn and wheat were recorded also. The effects of temperature on blight severity differed with the host. On corn, lesion development was not affected by temperature but plant growth was significantly reduced at the three intermediate temperature regimens. Average lesion size was significantly greater with strain C-71 than with strain 19860 at temperatures of 22/ 14 and 18/14 C but not at 30/ 26, 30/22, or 22/18 C. On oats, P. avenae strain significantly affected lesion development and disease was generally more severe at higher temperatures. On wheat, blight symptoms were generally more severe at higher temperatures but P. avenae strain had no significant effect. Under Georgia conditions, bacterial blight should be less severe in oats and wheat sown late in the fall season when the temperature is cooler. With corn, plant growth should be less reduced when the mean evening temperature is > 22 C. Strains of P. avenae should affect the severity of bacterial leaf blight of corn and oats but not of wheat. Bacterial leaf blight of corn, caused by Pseudomonas avenae (4), has been observed sporadically in corn fields in southern Georgia for several years. Severe outbreaks generally occur in late May and early June after periods of overcast and wet weather (7). Mean temperatures in south Georgia during this period are usually 18-24 C (7). The first symptoms are usually observed in early May when corn plants are 0.8-1 m tall. Although bacterial leaf blight of corn has been known for many years (2,8), little information is available on the effects of light and temperature on disease severity. Johnson et al (2) observed in a greenhouse that symptoms were abundant at 29-35 C, few at 24 C, and infrequent or lacking at 21 C. There are no reports of any observations on the effects of temperature on disease severity in oats and wheat. The objective of our study was to determine the influence of temperature and light on the severity of bacterial blight of corn, oats, and wheat. We wanted to determine if temperature and/ or light could also be used to predict disease occurrence. Experiments were done in environ