Antagonistic Activities of Epiphytic Bacteria from Soybean Leaves against Pseudomonas syringae pv. glycinea in vitro and in planta

Abstract Over two growing seasons, 273 bacterial strains were isolated from soybean leaves without and with bacterial blight symptoms caused by Pseudomonas syringae pv. glycinea (Psg). The majority of the isolates from leaves with symptoms were identified as Psg (43%), followed by Erwinia herbicola (21%), and Enterobacter/Erwinia (19%). The isolates from leaves without symptoms included mainly a group of unidentified Gram-negative bacteria (22%), Psg (21%), and E. herbicola (18%). Psg colonized the soybean leaves prior to saprophytic bacteria, and remained dominant during both seasons on healthy, as well as infected, leaves. Eighty-two saprophytic isolates were tested in vitro for their antagonistic activities against Psg, using an agar-diffusion assay. For the in planta assay, Psg and each isolate were simultaneously inoculated into wounds of pin-pricked leaves of greenhouse-grown soybean plants. Twenty-nine isolates were antagonistic in vitro. Nineteen isolates were able to suppress the growth of Psg and prevented the formation of leaf spots in planta when mixtures of isolate and pathogen were inoculated at ratios >1. Only 9 of the 82 isolates inhibited Psg in vitro as well as in planta. Most antagonists detected belonged to the genera Pseudomonas and the species Erwinia herbicola. The in planta assay should be a reliable predictor of field performance for screening of biological control agents. Received: 8 April 1996; Accepted: 22 October 1996     

Pectolytic enzymes produced by Pseudomonas syringae pv. glycinea

Abstract A comparative analysis of 16S ribosomal RNA sequences of all Peptostreptococcus species revealed that most members of the genus Peptostreptococcus should be divided into many different genera. The relationship between clostridia and peptostreptococci was analysed to find the phylogenetic position of peptostreptococci.     

Exopolysaccharides of the phytopathogen Pseudomonas syringae pv. glycinea.

Inhibition of DNA synthesis in Escherichia coli mutants in which the SOS-dependent division inhibitors SfiA and SfiC were unable to operate led to a partial arrest of cell division. This SOS-independent mechanism coupling DNA replication and cell division was characterized with respect to residual division, particle number, and DNA content. Whether DNA replication was blocked in the initiation or the elongation step, numerous normal-sized anucleate cells were produced (not minicells or filaments). Their production was used to evaluate the efficiency of this coupling mechanism, which seems to involve the cell division protein FtsZ (SulB), also known to be the target of the division inhibitors SfiA and SfiC. In the absence of DNA synthesis, the efficiency of coupling was modulated by the cyclic-AMP-cyclic-AMP receptor protein complex, which was required for anucleate cell production.     

Determination of Races of Pseudomonas syringae pv. glycinea Occurring in Europe

Fifty-eight strains of Pseudomonas syringae pv. glycinea were collected from France, Germany, Hungary, Italy, Poland, The Ukraine and the former Yugoslavia. The bacterial cultures were fluorescent on King's medium B, oxidase negative, produced levan, and induced a hypersensitive reaction (HR) on tobacco leaves within 24h. The race of each strain was determined by inoculating a set of seven differential soybean cultivars: Acme, Chippewa, Flambeau, Harosoy, Lindarin, Merit and Norchief.Conditions of plant cultivation, bacterial inoculation and plant incubation had to be standardized scrupulously to obtain reproducible results. Authentic strains belonging to races 1, 4, 5, and 6 produced the expected reactions on the differentials. However, strains of race 2 and race 4 induced identical responses on the differential cultivars. The differentiating critierion between these two races was the ability of race 2 to produce a brown diffusable pigment on King's medium B. The most prevalent race, occurring in every European country studied, was race 4. This is the most aggressive race of P. glycinea, since it infects all the cultivars of the set of differentials. From 58 strains tested, 42 belonged to race 4, 4 to race 6, and 6 to race 9. For two strains race identification was impossible. The remaining 4 isolates did not fit into the pattern of known races. It is proposed that these strains belong to a new race (no. 10) which is similar to race 5, but can inf, ect the soybean cultivar ‘Lindarin’. On the other hand, race 10 can not infect cv. ‘Chippewa’, in contrast to race 9.     

Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

Comparison of the Behavior of Epiphytic Fitness Mutants of Pseudomonas syringae under Controlled and Field Conditions

The epiphytic fitness of four Tn5 mutants of Pseudomonas syringae that exhibited reduced epiphytic fitness in the laboratory was evaluated under field conditions. The mutants differed more from the parental strain under field conditions than under laboratory conditions in their survival immediately following inoculation onto bean leaves and in the size of the epiphytic populations that they established, demonstrating that their fitness was reduced more under field conditions than in the laboratory. Under both conditions, the four mutants exhibited distinctive behaviors. One mutant exhibited particularly large population decreases and short half-lives following inoculation but grew epiphytically at near-wild-type rates, while the others exhibited reduced survival only in the warmest, driest conditions tested and grew epiphytically at reduced rates or, in the case of one mutant, not at all. The presence of the parental strain, B728a, did not influence the survival or growth of three of the mutants under field conditions; however, one mutant, an auxotroph, established larger populations in the presence of B728a than in its absence, possibly because of cross-feeding by B728a in planta. Experiments with B728a demonstrated that established epiphytic populations survived exposure of leaves to dry conditions better than newly inoculated cells did and that epiphytic survival was not dependent on the cell density in the inoculum. Three of the mutants behaved similarly to two nonpathogenic strains of P. syringae, suggesting that the mutants may be altered in traits that are missing or poorly expressed in naturally occurring nonpathogenic epiphytes.     

Characterization and expression of two avirulence genes cloned from Pseudomonas syringae pv. glycinea.

Two avirulence genes, avrB and avrC, from race 0 of Pseudomonas syringae pv. glycinea, were sequenced and found to encode single protein products of 36 and 39 kilodaltons, respectively. The proteins had neither recognizable signal peptide sequences nor significant stretches of hydrophobic amino acids that might indicate membrane association. Both avrB and avrC had relatively low position 3 and overall G+C contents, which suggests that they may have been recently introduced into P. syringae pv. glycinea. The deduced amino acid sequences of the proteins encoded by avrB and avrC shared 42% identical amino acids. However, when introduced into race 4 of P. syringae pv. glycinea, each gene directed a unique pattern of hypersensitive reactions on several differential soybean cultivars. The avrC protein was overproduced in Escherichia coli cells and deposited as insoluble inclusion bodies in the cell cytoplasm. The avrC protein could be solubilized with urea-octyl glucoside treatment, but neither the solubilized protein nor the intact inclusion bodies elicited a hypersensitive reaction in soybean leaves.     

丁香假单胞大豆致病变种两个III型效应因子的克隆及功能分析

为了研究Ⅲ型泌出效应因子在丁香假单胞大豆致病变种中的作用,利用反向PCR技术,首次从丁香假单胞大豆致病变种全基因组中克隆得到两个效应因子HopAB1和HopAF1基因的同源物,分别命名为HopAB1s和HopAF1s。生物信息学分析表明,HopAB1s基因全长是1 572 bp,编码523个氨基酸;HopAF1s基因全长是855 bp,编码284个氨基酸。即基因的登录号分别为JF826562和JF826563。保守功能区预测显示HopAB1s在N末端包含一个E3泛素连接酶功能区。将这2个基因克隆到PVX二元表达载体并转化农杆菌,利用农杆菌介导的瞬时侵染技术在本生烟中表达,发现2个效应因子均能抑制由鼠凋亡因子激发的细胞程序性死亡;将烟草疫霉接种在表达效应基因的区域,发现效应因子能促进烟草疫霉侵染烟草,因此本研究得到的两个效应因子是免疫抑制因子,为进一步研究该菌的致病机理奠定基础。     

Chemotaxis by Pseudomonas syringae pv. tomato

Optimal laboratory conditions for studying chemotaxis by Pseudomonas syringae pv. tomato were determined by using the Adler capillary tube assay. Although they are not an absolute requirement for chemotaxis, the presence of 0.1 mM EDTA and 1 mM MgCl₂ in the chemotaxis buffer (10 mM potassium phosphate [pH 7.2]) significantly enhanced the response to attractant. The addition of mannitol as an energy source had little effect. The optimal temperature for chemotaxis was 23°C, which is 5°C below the optimal growth temperature for this pathogen. The best response occurred when the bacteria were exposed to attractant for 60 min at a concentration of approximately 5 × 10⁶ CFU/ml. P. syringae pv. tomato was strongly attracted to citric and malic acids, which are the predominant organic acids in tomato fruit. With the exception of asparagine, the major amino acids of tomatoes were weak to moderate attractants. Glucose and fructose, which account for approximately 47% of tomato dry matter, also elicited poor responses. In assays with tomato intercellular fluid and leaf surface water, the bacterial speck pathogen could not chemotactically distinguish between a resistant and a susceptible cultivar of tomato.     

Flagellin glycosylation island in Pseudomonas syringae pv. glycinea and its role in host specificity

The deduced amino acid sequences of the flagellins of Pseudomonas syringae pv. tabaci and P. syringae pv. glycinea are identical; however, their abilities to induce a hypersensitive reaction are clearly different. The reason for the difference seems to depend on the posttranslational modification of the flagellins. To investigate the role of this posttranslational modification in the interactions between plants and bacterial pathogens, we isolated genes that are potentially involved in the posttranslational modification of flagellin in P. syringae pv. glycinea (glycosylation island); then defective mutants with mutations in these genes were generated. There are three open reading frames in the glycosylation island, designated orf1, orf2, and orf3. orf1 and orf2 encode putative glycosyltransferases, and mutants with defects in these open reading frames, deltaorf1 and deltaorf2, secreted nonglycosylated and slightly glycosylated flagellins, respectively. Inoculation tests performed with these mutants and original nonhost tobacco leaves revealed that deltaorf1 and deltaorf2 could grow on tobacco leaves and caused symptom-like changes. In contrast, these mutants failed to cause symptoms on original host soybean leaves. These data indicate that putative glycosyltransferases encoded in the flagellin glycosylation island are strongly involved in recognition by plants and could be the specific determinants of compatibility between phytopathogenic bacteria and plant species.     

Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.     

Population Dynamics of Pseudomonas syringae pv. tomato Strains on Tomato Cultivars Rio Grande and Rio Grande-Pto under Field Conditions

We examined the effects of the Pto resistance locus on the population dynamics of Pseudomonas syringae pv. tomato (Pst) strains in field experiments with the nearly isogenic tomato lines Rio Grande (RG, susceptible to Pst races 0 and 1) and Rio Grande-Pto (RG-Pto, resistant to Pst race 0, susceptible to Pst race 1). Pst strain SM78-1Sm^r (race 0) grew well under field conditions and caused ample bacterial speck disease on susceptible RG plants. In contrast, strain DC3000 failed to establish large populations when inoculated onto field grown RG plants. Mean population sizes of SM78-1Sm^r were 4–5 orders of magnitude larger on RG than RG-Pto plants indicating that RG-Pto plants were highly effective in attenuating pathogen population development. Most of the sampled leaflets from RG-Pto field plots harboured small numbers of SM78-1Sm^r. However, population sizes SM78-1Sm^r as large as 10⁵–10⁶ CFU were found on a few leaflets. Bacteria isolated from these leaflets had phenotypes characteristic of Pst race 1 strains. In growth chamber plant assays, the bacterial strains grew well and caused typical speck lesions on RG-Pto plants. The strains appeared to be race-shift mutants of SM SM78-1Sm^r. Interestingly, results from DNA hybridization experiments demonstrated that the race-shift mutants were deleted for the avirulence gene, avrPto but not for avrPtoB.     

Sugar-specific Attachment of Pseudomonas syringae pv. glycinea to Isolated Single Leaf Cells of Resistant Soybean Cultivars

Phase-contrast and scanning electron microscopy showed races of P. synngae pv. glycinea uniformly distributed over and attached to the whole surface of isolated single leaf cells of resistant soybean cultivars, as early as 30 to 180 min after inoculation. On the contrary, attachment in the compatible interaction did not occur within 10—15 h. In a later period, compatibility was characterized by the formation of adherent bacterial clusters. Early attachment of races 1 and 6 to cv. Harosoy and that of race 5 to cv. Flambeau leaf cells, each representing incompatible interaction, could be inhibited by L-rhamnose and D-glucose, respectively. Furthermore, the lack of Mn²⁺ and Fe²⁺ and heat-treatment of plant cells also affected the early attachment in incompatible combinations and resulted in cluster formation, suggesting incompatibility rather than compatibility to be the active phenomenon. Pre-inoculation of cells with an incompatible race induced changes that caused compatible bacteria also to distributively attach to plant cell surface indicating that a transfer of information or surface alterations occur upon attachment in incompatible interaction.     

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.     

Contribution of the Regulatory Gene lemA to Field Fitness of Pseudomonas syringae pv. syringae

In Pseudomonas syringae pv. syringae, lemA is required for brown spot lesion formation on snap bean and for production of syringomycin and extracellular proteases (E. M. Hrabak and D. K. Willis, J. Bacteriol. 174: 3011-3022, 1992; E. M. Hrabak and D. K. Willis, Mol. Plant-Microbe Interact. 6:368-375, 1993; D. K. Willis, E. M. Hrabak, J. J. Rich, T. M. Barta, S. E. Lindow, and N. J. Panopoulos, Mol. Plant-Microbe Interact. 3:149-156, 1990). The lemA mutant NPS3136 (lemA1::Tn5) was previously found to be indistinguishable from its pathogenic parent B728a in its ability to grow when infiltrated into bean leaves of plants maintained under controlled environmental conditions (Willis et al., Mol. Plant-Microbe Interact. 3:149-156, 1990). We compared population sizes of NPS3136 and B728aN (a Nal(supr) clone of wild-type B728a) in two field experiments to determine the effect of inactivation of lemA on the fitness of P. syringae pv. syringae. In one experiment, the bacterial strains were spray inoculated onto the foliage of 25-day-old bean plants. In the other, seeds were inoculated at the time of planting. In both experiments, the strains were inoculated individually and coinoculated in a 1:1 ratio. NPS3136 and B728aN achieved similar large population sizes on germinating seeds. However, in association with leaves, population sizes of NPS3136 were diminished relative to those of B728aN in both experiments. Thus, lemA contributed significantly to the fitness of P. syringae pv. syringae in association with bean leaves but not on germinating seeds under field conditions. When NPS3136 was coinoculated with B728aN, the mutant behaved as it did when inoculated alone. However, population sizes of B728aN in the coinoculation treatment were much lower than those when it was inoculated alone. Inactivation of the lemA gene appeared to have rendered the mutant suppressive to B728aN.