Inferring the evolutionary history of the plant pathogen Pseudomonas syringae from its biogeography in headwaters of rivers in North America, Europe, and New Zealand.

Nonhost environmental reservoirs of pathogens play key roles in their evolutionary ecology and in particular in the evolution of pathogenicity. In light of recent reports of the plant pathogen Pseudomonas syringae in pristine waters outside agricultural regions and its dissemination via the water cycle, we have examined the genetic and phenotypic diversity, population structure, and biogeography of P. syringae from headwaters of rivers on three continents and their phylogenetic relationship to strains from crops. A collection of 236 strains from 11 sites in the United States, in France, and in New Zealand was characterized for genetic diversity based on housekeeping gene sequences and for phenotypic diversity based on measures of pathogenicity and ice nucleation activity. Phylogenetic analyses revealed several new genetic clades from water. The genetic structure of P. syringae populations was not influenced by geographic location or water chemistry, whereas the phenotypic structure was affected by these parameters. Comparison with strains from crops revealed that the metapopulation of P. syringae is structured into three genetic ecotypes: a crop-specific type, a water-specific type, and an abundant ecotype found in both habitats. Aggressiveness of strains was significantly and positively correlated with ice nucleation activity. Furthermore, the ubiquitous genotypes were the most aggressive, on average. The abundance and diversity in water relative to crops suggest that adaptation to the freshwater habitat has played a nonnegligible role in the evolutionary history of P. syringae. We discuss how adaptation to the water cycle is linked to the epidemiological success of this plant pathogen.

Puccinia acroptili on Russian Knapweed in Colorado, Montana, and Wyoming.

Acroptilon repens (L.) DC. (Russian knapweed) is a long-lived perennial weed from central Asia that is widely distributed in the western United States (U.S.). Recently, accessions of a rust disease were collected from Colorado (CO), Montana (MT), and Wyoming (WY) for comparison with Eurasian isolates. U.S. accessions had two-celled teliospores with slight constrictions in the middle and urediniospores with three germ pores ± equatorial in location. Urediniospores were (state, width × length, [n = 100]): CO, 16.4 to 25.7 × 19.2 to 27.0 µm; MT, 18.4 to 23.1 × 17.4 to 24.6 µm; and WY, 18.0 to 26.2 × 20.2 to 26.7 µm. These were similar to those of 16.6 to 25.7 × 21.2 to 28.0 µm from two New Mexican (NM) herbarium specimens (BPI Nos. 1107952 and 1110177) (1). Teliospores measured 19.9 to 27.7 × 29.8 to 47.4 µm, 17.4 to 26.0 × 32.4 to 44.2 µm, 16.5 to 27.5 × 29.4 to 45.7 µm, and 18.7 to 27.6 × 31.0 to 46.4 µm for CO, MT, WY, and NM accessions, respectively. These rust isolates have been identified as Puccinia acroptili Syd. on the basis of host plant record and spore morphology (2). To our knowledge, this is the first record of P. acroptili in CO, MT, and WY. Besides NM, P. acroptili has been reported in North America from California, British Columbia, and Saskatchewan. References: (1) M. E. Palm and S. G. Vesper. Plant Dis. 75:1075, 1991. (2) D. B. O. Savile. Can. J. Bot. 48:1567, 1970.

Biological Control of Pseudomonas syringae pv. syringae, the Causal Agent of Basal Kernel Blight of Barley, by Antagonistic Pantoea agglomerans.

ABSTRACT Strains of Pantoea agglomerans (synanamorph Erwinia herbicola) suppressed the development of basal kernel blight of barley, caused by Pseudomonas syringae pv. syringae, when applied to heads prior to the Pseudomonas syringae pv. syringae infection window at the soft dough stage of kernel development. Field experiments in 1994 and 1995 revealed 45 to 74% kernel blight disease reduction, whereas glasshouse studies resulted in 50 to 100% disease control depending on the isolate used and barley cultivar screened. The efficacy of biocontrol strains was affected by time and rate of application. Percentage of kernels infected decreased significantly when P. agglomerans was applied before pathogen inoculation, but not when coinoculated. A single P. agglomerans application 3 days prior to the pathogen inoculation was sufficient to provide control since populations of about 10(7) CFU per kernel were established consistently, while Pseudomonas syringae pv. syringae populations dropped 100-fold to 2.0 x 10(4) CFU per kernel. An application to the flag leaf at EC 49 (before heading) also reduced kernel infection percentages significantly. Basal blight decreased with increasing concentrations (10(3) to 10(7) CFU/ml) of P. agglomerans, with 10(7) CFU/ml providing the best control. For long-term preservation and marketability, the survival of bacterial antagonists in several wettable powder formulations was tested. Over all formulations tested, the survival declined between 10- to >100-fold over a period of 1.5 years (r = -0.7; P = 0.000). Although not significant, storage of most formulations at 4 degrees C was better for viability (90 to 93% survival) than was storage at 22 degrees C (73 to 79%). However, long-term preservation had no adverse effect on biocontrol efficacy.

Characterization of a Vascular Wilt of Erythroxylum coca Caused by Fusarium oxysporum f. sp. erythroxyli Forma Specialis Nova.

A new forma specialis of Fusarium oxysporum (F. oxysporum f. sp. erythroxyli) pathogenic to Erythroxylum coca and E. novogranatense is described. The pathogen was isolated from the vascular tissue of diseased plants from an Erythroxylum plantation in Hawaii. This pathogen causes vascular wilt symptoms and death in both E. coca and E. novogranatense plants as soon as 7 weeks after soil infestation. The pathogenicity of seven isolates from the affected field was determined in field and growth-chamber studies. Genetic variation was not detected among the seven Hawaiian isolates, using arbitrarily primed polymerase chain reaction. The seven isolates could be differentiated from a strain isolated from a diseased E. coca plant from South America. All Hawaiian isolates and the South American isolate belonged to a single vegetative compatibility group.

Epiphytic populations of Pseudomonas syringae on barley.

The epiphytic populations of Pseudomonas syringae were monitored on 23 barley entries planted in the field in four replications during the summer of 1986, and on six selected entries during the summer of 1987, from the second-leaf stage until senescence. Populations were initially low (0-3 log colony-forming units (cfu) per leaf) in all but one entry; they generally increased throughout the season, and at the end they reached 3-7 log cfu/leaf. Significant differences among the average epiphytic populations were found in the 1986 trial; only one entry, however, had a significantly different average population in the 1987 trial. The slopes of population increase were also compared: significant differences were observed in 1986 but not in 1987. In addition to epiphytic population counts, the percentage of ice nucleation active bacteria was determined in the population isolated from each leaf sample, and averaged throughout the season for each entry. Significant differences were observed in 1986 and in 1987. When the entries were ranked according to their average epiphytic population and compared between the two experiments, they were found to be very similar. The same was not true for the other parameters studied in the experiment.

Identification and characterization of rhizosphere-competent bacteria of wheat.

To obtain rhizosphere-competent bacteria which could subsequently be modified for the development of biological control agents, bacteria were isolated from the rhizosphere and rhizoplane of wheat and barley plants by standard techniques. Of these isolates, 60 were selected for field testing as spring wheat seed inoculants in 1985. Isolates were marked genetically for resistance to antibiotics via selection of spontaneous mutants to detect and monitor isolates in the field. Forty-three days after planting, the average log(10) CFU/mg (dry weight) of roots and rhizosphere soil for the mutant isolates sampled ranged from 0 to 3.4. Twenty mutant isolates were retested in 1986. A total of 4 isolates were not detected, but the other 16 had an average root colonization value of log(10) 2.1 CFU and a range of log(10) 0.9 CFU to log(10) 3.2 CFU when sampled 32 days after planting. The average colonization value dropped to log(10) 1.1 CFU 51 days later. Some isolates detected previously were not detected in the second sampling; others had root colonization values similar to those obtained in the first sampling. Mutant isolates of rhizosphere bacteria included Bacillus pumilus, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces spp., Xanthomonas maltophilia, and a saprophytic coryneform. Mixtures of isolates from different genera and species were compatible on seeds and roots.

A microbiological approach to nutrition.

Fermentation is known to modify nutritional value of foods positively. A new technique for supplementing traditional fermented foods with limiting amino acids is presented. Normal fermentative bacteria are induced to produce specific amino acids during the fermentation period. Acceptable food products have been prepared using fermented grain. The concept may be applicable to developing countries.

Dietary selection for lysine by the chick.

Broiler chicks were provided choices of synthetic diets (a) adequate or low in lysine, and (b) adequate in or devoid of lysine. In each case, chicks consumed some of each diet offered, but preference was shown for the adequate lysine diet. Growth rates of chicks given choices ranged from 80% of that of chicks fed an adequate lysine diet with no choice for two weeks, then growth rates fell to about 60% of those fed adequate lysine. In another study, chicks were fed a diet devoid of lysine but were offered pure L-lysine HCl in a separate feeder. These chicks selected some of the supplementary lysine, but their body weights were only 68% of the body weight of chicks fed an adequate lysine diet after 21 days. Chicks given a choice of diets prepared with an adequate quantity of either L- or D-lysine preferred with L-lysine diet but did not select sufficient quantity to reach normal growth. These observations indicate that chicks can discern the presence of L-lysine in diets or separately, but will not select sufficient quantity for maximum growth potential. A diet prepared with D-lysine was more acceptable than one completely devoid of lysine, suggesting some sensory recognition for lysine.

Effects of hydroclytic enzymes on plant-parasitic nematodes.

Proteases, lipase, and chitinase killed Tylenchorhynchus dubius in vitro and in soil. Tylenchorhynchus dubius was more susceptible to the enzymes than Pratylenchus penetrans. Papain was the most effective protease, and other enzymes were less effective. Heating enzymes to 80 C for 10 min greatly reduced nematicidal effectiveness. Scanning electron micrographs showed that papain and chitinase produced structural changes in the cuticle of T. dubius. Lipase removed a thin outer layer. Papain removed material filling the striata, or furrow, between the horizontal bands. When added to soil, chitinase, lipase, collagenase, and proteases (papain and bromelain) decreased motility of T. dubius populations up to 75%. Bromelain was the most active in soil against T. dubius, and collagenase was the most active in soil against P. penetrans.

Use of activated charcoal for the removal of patulin from cider.

Penicillium urticae (NRRL 2159A) was grown in culture broth containing 1 muCi of [1-14C-A1acetate to produce [14C]patulin. [14C]patulin was purified from the broth and added to apple cider. After the patulin concentration of the cider was adjusted to 30 mug/ml with unlabeled patulin, the cider was subjected to various charcoal treatments. [14C]patulin was completely removed by shaking the cider with 20 mg of activated charcoal per ml and by eluting the cider through a 40- to 60-mesh charcoal column. Activated charcola at 5 mg/ml reduced patulin in naturally contaminated cider to nondetectable levels.

Effect of Glucose and Adenosine Phosphates on Production of Extracellular Carbohydrases of Alternaria solani.

Production of carbohydrases by Alternaria solani is inhibited by glucose under low growth conditions. In an enriched medium, glucose has little effect on the production of polygalacturonase and cellulase while it still suppresses production of beta-glucosidase. Low levels of all three enzymes were produced in the absence of their respective substrates. Such regulation has been found with many organisms. However, far greater production of these carbohydrases occurred with additions of adenosine phosphates to the growth media. Highest stimulation of enzyme production was by adenosine 5'-phosphate. Adenosine 5'-triphosphate and cyclic 3', 5'-adenosine monophosphate gave lesser amounts. Starvation appears to induce production of extracellular carbohydrases and adenosine 5'-phosphate may have a role in the starvation process.

Selecting lysine-excreting mutants of lactobacilli for use in food and feed enrichment.

Lysine analogues are used to select for lysine-excreting mutants of Lactobacillus plantarum. The use of lactobacilli that excrete lysine for the enrichment of foods and feedstuffs by fermentation is discussed. The increase in lysine content of soybean milk by a mutant of L. bulgaricus and in silage by L. plantarum is shown.

Improved solid medium for the detection and enumeration of pectolytic bacteria.

An improved solid agar medium (MP medium) has been developed which allows detection of pectolytic activity in bacteria. Organisms tested exhibited a variety of regulatory controls governing pectate lyase synthesis. The medium contains mineral salts, pectin, and yeast extract. After growth of the organisms, the agar plate is flooded with a polysaccharide precipitant, and pectolytic activity is shown by clear zones around active colonies. High concentrations of phosphate are shown to be necessary for pectic enzyme formation on solid media. The medium has successfully been used to detect pectolytic organisms in soil, forest litter, and rotting vegetable samples.

Isolation of fluorescent pseudomonads with a selective medium.

Incorporation of novobiocin, penicillin, and cycloheximide into a standard medium for fluorescence selects for fluorescent pathogenic and free-living pseudomonads.

Taxonomy of phytopathogenic pseudomonads.

Phytopathogenic pseudomonads were placed into four major groups on the basis of nutritional and physiological characteristics. Group I consists of 86 strains of phytopathogens distinguishable from other fluorescent pseudomonads by low growth rates, ability to induce hypersensitivity on tobacco, absence of arginine dihydrolase, and relatively limited ranges of carbon sources. Most of these strains cannot utilize benzoate, 2-ketogluconate, spermine, beta-alanine, l-isoleucine, l-valine, and l-lysine. Most of the organisms in group I clustered into a small number of subgroups, each of which generally corresponded to a previously recognized nomenspecies. These subgroups differ with respect to the number of substrates used. As a rule, the organisms that utilize the fewest substrates have the most limited host ranges. The fluorescent pseudomonads of group II are arginine dihydrolase-positive and utilize a considerably larger number of carbon sources. Most pathogens of group II are similar to Pseudomonas fluorescens biotype A. Groups III and IV consist of nonfluorescent pseudomonads. These two groups can be distinguished by the number of carbon sources used and by pigmentation. An amended description of the flurescent pseudomonads and their internal subdivision is presented.