STUDIES IN THE BACTERIAL DISEASES OF SUDAN CROPS

The atypical symptoms first described by Bryan (1932) of the angular leaf spot disease of cotton caused by Xanthomonas malvacearum (E. F. Sm.) Dowson were reproduced by inoculation into seeds, stem apices or buds. The lesions that developed on the veins of the newly produced leaves were elongated and water-soaked, becoming dark brown. The leaf tissue dependent upon infected veins became yellow, flaccid and withered. The development of these symptoms was enhanced when inoculations were made into opening buds or germinating seeds as compared with inoculations into closed buds or dormant seeds.
In other bacterial diseases caused by Xanthomonas spp., somewhat atypical symptoms could also be produced by bud inoculation into the appropriate host. Those produced by X. ricini (Yoshi & Takimato) Dowson on castor, closely resembled the vein lesions described above on cotton but resulted only from bud inoculations; inoculations into stem apices and seeds failed to produce them. In dolichos bean inoculated with X. phaseoli (Smith) Dowson, atypical symptoms were produced only by seed inoculations and were confined to the first simple leaves (prophylls).
The differences in the production of atypical symptoms on the three hosts are correlated with differences in host structure and with the degree of virulence of the pathogen. The leaf parasite X. ricini , for example, which cannot infect castor bean stems, does not produce atypical symptoms when inoculated below the stem apex.
From the data discussed below, the incidence of atypical symptoms is attributed to infection either of an actively growing tissue or of a telescoped structure which subsequently completes its development.
The atypical symptoms of the cotton disease are not caused by a special strain of X. malvacearum. Further, they are not a peculiarity of this disease but may also develop in other necrotic diseases under similar conditions.     

STUDIES IN THE BACTERIAL DISEASES OF SUDAN CROPS

The bacterial leaf spot disease of jute ( Corchorus olitorius ) is characterized by the development of brown circular spots, about 2–5 mm. in diameter, on the leaves, surrounded, sometimes, by a yellow halo. The disease on the stems causes elongated lesions, and on the capsules produces small sunken spots. Defoliation and death of the stems may result in severe infections. The disease is ascribed to a new variety of Xanthomonas nakatae (Okabe) Dowson, the cause of a somewhat similar disease of Corchorus capsularis in Formosa (Elliott, 1951), on the ground that: (i) the minor differences between the two pathogens in biochemical characters are considered well within strain differences, and (ii) the Sudan pathogen does not infect C. capsularis , and Xanthomonas nakatae has not been reported to infect Corchorus olitorius . These differences do not justify the creation of a new species. The name Xanthomonas nakatae v. olitorii is suggested for the cause of the disease under consideration.     

Biocidal activity in plant pathogenic Acidovorax, Burkholderia, Herbaspirillum, Ralstonia and Xanthomonas spp.

Antibacterial and antifungal activity was investigated for strains of Acidovorax spp., Burkholderia spp., Herbaspirillum rubrisubalbicans and Ralstonia solanacearum ; strains representing 118 species and pathovars of Xanthomonas were also tested for phytotoxic capacity. Antibacterial activity was present in all Burkholderia spp. except B. andropogonis , in biovars II and III of R. solanacearum but not in biovars I and IV, and in two strains of Xanthomonas. Little antibacterial activity was recorded for Acidovorax spp. Antifungal activity was expressed by most strains of A. avenae ssp. avenae and A. avenae ssp. cattleyae. Weak or variable antifungal reactions were given by strains of A. avenae ssp. citrulli and no activity was expressed by A. konjaci. Most strains of B. caryophylli, B. cepacia, B. gladioli pv. agaricicola, B. gladioli pv. alliicola, B. gladioli pv. gladioli , B. glumae and B. plantari produced extensive inhibition zones against Rhodotorula mucilaginosa. Strains of H. rubrisubalbicans and R. solanacearum gave negative, weak or variable reactions. Strains of Xanthomonas spp. exhibited no antifungal activity. In all cases antifungal activity was caused by a low molecular weight toxin. Three Xanthomonas strains exhibited phytotoxic activity. The ecological implications of these data are discussed.     

Repeat domain diversity of avrBs3-like genes in Ralstonia solanacearum strains and association with host preferences in the field

Genes homologous to avrBs3 of Xanthomonas were detected in 309 strains of Ralstonia solanacearum biovars 3, 4, and 5 but not biovar 1 or 2. A statistically significant association between the originating plant species and internal repeats of the gene was found. Sequences of repeats and variation between nearly clonal strains revealed evidence of frequent recombination.     

BACTERIAL DISEASES OF STONE-FRUIT TREES IN BRITAIN

A bacterial shoot wilt, observed at the East Malling Research Station in 1923 and 1924, is described.
A bacterium isolated from infected shoots has been shown by inoculation experiments to be the cause of the disease.
The organism not only infects young green shoots, but experiments have shown that it can also cause leaf spots, and that it can induce gummosis and cankers on woody branches and on stems.
Cultural studies of the organism suggest that it has not previously been described, so the name Ps. prunicola is proposed for it.     

STUDIES IN THE BACTERIAL DIE-BACK AND CANKER DISEASE OF POPLAR

The bacterial die-back and canker of poplar is characterized by dark necrotic areas in the leaves, die-back accompanied by cracks in the bark of 1–4-year-old branches, and by cankers on the trunk and branches of different ages. The cankers may be 'closed' or 'open' and when moist exude slime containing bacteria.
The disease is caused by Pseudomonas syringae f. sp. populea , a new forma specialis closely resembling Ps. syringae in bacteriological characters but differing from it in parasitism. The association of the slime with the bacteria is essential for the production of typical lesions.
Inoculations with the pathogen alone, or in association with other organisms produce atypical symptoms.
The bacteria attack the cortical parenchyma and occasionally invade the xylem vessels.     

PopF1 and PopF2, two proteins secreted by the type III protein secretion system of Ralstonia solanacearum, are translocators belonging to the HrpF/NopX family

Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum.     

BACTERIAL CANKER OF STONE-FRUITS III. INOCULUM CONCENTRATION AND TIME OF INOCULATION IN RELATION TO LEAF-SCAR INFECTION OF CHERRY

Leaf scars on the fruiting spurs of the cherry varieties Roundel (high resistance) and Napoleon (low resistance) were inoculated with Pseudomonas mors-prunorum on four separate occasions in the autumn, using, on each occasion, the same range of five different inoculum concentrations. The results, recorded the following year, showed that the percentage diseased spurs (disease incidence) and the severity of the disease symptoms (disease severity) both increased with inoculum concentration.
The median threshold concentration of inoculum (T. C. 50), defined as the concentration necessary to give 50% diseased spurs, varied with time of inoculation, but on all occasions was considerably higher for Roundel than for Napoleon.
In another experiment the leaf scars at the nodes of the current year's growth, inoculated at weekly intervals throughout the autumn, were found to be susceptible from the beginning of September to the latter part of October. During this period disease incidence varied considerably with time of inoculation. There was evidence that this variation was related to two factors which influenced the numbers of bacteria penetrating into leaf scars, namely, (1) the rate of evaporation of the infection drop, and (2) the rate of suction of inoculum into the vessels of the leaf traces.
The experiments provided evidence of a long infection period beginning early in the autumn. It is suggested, therefore, that the timing of bactericidal sprays in the autumn be advanced and that the present concept of 'protective' sprays in disease control be replaced by one based on the eradication of external sources of inoculum.     

Classification of plant associated bacteria using RIF, a computationally derived DNA marker

A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.     

hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria.

The majority of bacterial plant diseases are caused by members of three bacterial genera, Pseudomonas, Xanthomonas, and Erwinia. The identification and characterization of mutants that have lost the abilities to provoke disease symptoms on a compatible host and to induce a defensive hypersensitive reaction (HR) on an incompatible host have led to the discovery of clusters of hrp genes (hypersensitive reaction and pathogenicity) in phytopathogenic bacteria from each of these genera. Here, we report that predicted protein sequences of three hrp genes from Pseudomonas solanacearum show remarkable sequence similarity to key virulence determinants of animal pathogenic bacteria of the genus Yersinia. We also demonstrate DNA homologies between P. solanacearum hrp genes and hrp gene clusters of P. syringae pv. phaseolicola, Xanthomonas campestris pv. campestris, and Erwinia amylovora. By comparing the role of the Yersinia determinants in the control of the extracellular production of proteins required for pathogenicity, we propose that hrp genes code for an export system that might be conserved among many diverse bacterial pathogens of plants and animals but that is distinct from the general export pathway.     

BACTERIAL ROTTING OF APPLE FRUIT

Three bacterial isolates obtained from a rotten apple were found to produce actively spreading firm brown rots when stab-inoculated into Bramley's Seedling apples, but bacteria were not found at the front of the rot.
One isolate was a capsulated, non-motile, non-fluorescent, Gram-negative rod apparently not described previously. The name proposed for the isolate is Pseudomonas pomi. The other two isolates were the same and were non-capsulated, non-fluorescent, Gram-negative motile rods, usually with two polar flagella, and were considered to be identical with Ps. melophthora Allen & Riker (Allen & Riker, 1932).
The three isolates grew on media at pH 5.0 and in apples with juice of pH 3.5. For each isolate the square of the distance of the front of the rot from the point of inoculation of apples, was found to be directly proportional to the time after inoculation. The isolates were able to invade fruit at the stalk and eye ends and where the skin had been pricked. No apparent infection followed spraying leafy shoots or flowers of Bramley's Seedling trees with bacterial suspensions and there was no visible reaction after hypodermic inoculation of bacterial suspensions into apple shoots.     

The specificity of an immune serum to the exocellular lipopolysaccharide of Pseudomonas wieringae

The serum obtained to exocellular lipopolysaccharide (ELPS) of Pseudomonas wieringae selectively agglutinated strains of pathovar of P. syringae and did not agglutinated strains of P. cichorii, P. solanacearum, P. gladioli pv. allicola, P. fluoroviolaceus, strains of nonphytopathogenic pseudomonads as well as bacteria of the genera Erwinia, Bacillus, Xanthomonas, Klebsiella. Consequently, the antigen determinant common with antigen of the species Pseudomonas syringae is present in the composition of ELPS.     

AM真菌对青枯菌和根际细菌群落结构的影响

利用传统的平板培养与DGGE相结合的技术手段,研究了接种AM真菌对番茄根际土壤中的青枯菌和细菌群落结构的影响。结果表明,菌根根际土壤中的细菌总量和总DNA量都高于非菌根根际土壤,其中前者的青枯菌种群数量比后者低60倍;DGGE图谱也证实了AM真菌对青枯菌的抑制效应,还揭示出接种AM真菌对根际土壤中细菌群落结构所产生的复杂的影响。文章对AM真菌抑制青枯菌的机制进行了探讨。     

大豆斑疹病菌harpin编码基因的克隆与特性研究

根据黄单胞菌harpin编码基因的同源性,设计简并引物,采用PCR方法从大豆斑疹病菌（Xanthomonas axonopodis pv.glycines, Xag）中克隆了402 bp的[STBX]hpa1[STBZ]同源基因,构建于表达载体pET30(a)上经转化大肠杆菌BL21菌株,获得基因工程菌BHR_3。基因工程菌诱导表达后经收集菌体和破碎细胞,得到表达产物为151kD的蛋白质。该蛋白质富含甘氨酸,不含半胱氨酸,对热稳定,对蛋白酶K敏感,可在非寄主烟草上激发过敏反应。激发的过敏反应需要植物体内水杨酸的积累,还可被真核生物代谢抑制剂抑制。序列比较显示,该基因与Xag中hpaG基因相同,与其它黄单胞菌中的hpa1基因有51.4%～93.8%的同源性,与其它革兰氏阴性植物病原细菌的harpin编码基因无同源性。据此把该基因产物鉴定为harpinXag。黄单胞菌harpin蛋白质序列比较发现,GG_GGG基序的多少并不是harpin蛋白的唯一特性。这为利用harpin蛋白开展植物病害控制的基因药物学设计提供了科学线索。     

Inhibition of hydrolytic enzyme activities and plant pathogen growth by invertase inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered.

A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.     

SOME OBSERVATIONS ON THE BACTERIAL CONTENT OF WATER IN WATERCRESS BEDS

SUMMARY: Samples of water flowing into watercress beds had higher bacterial contents during the summer months although this was less marked among samples from deep underground sources. Outlet samples showed higher contents than inlet samples and the bacterial content varied markedly with season, being highest in summer.
The presumptive coli-aerogenes contents at 30°, 37° and 44° also showed a similar seasonal variation among samples from the outlets but the marked inferiority of samples other than from bores was no longer apparent after the water had flowed through the beds. The dominant types were Bact. aerogenes type I, pectate liquefiers, Bact. coli type I, and Intermediate type I.
The importance of the pectate liquefiers was confirmed by plating on pectate gel and by their isolation from these plates and from MacConkey's broth.     

Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps(-) mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps(-) mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps(-) mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.     

Inhibition of Hydrolytic Enzyme Activities and Plant Pathogen Growth by Invertase Inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, α - l -arabinofuranosidase and β -glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.