Bacterial Stem Rot in Greenhouse Pepper (Capsicum annuum L.) in Sardinia (Italy): Occurrence of Erwinia carotovora subsp. carotovora

An unusual bacterial disease was observed in pepper plants during research carried out in greenhouses in central‐north Sardinia. The characteristics were: the presence of lesions and exudates on stems, soft rot of the pith, and a brownish‐black colour in the petioles and leaf‐veins. Only two isolates of 21 were pathogens. One was obtained from exudate present on the stem and the other from pith. Experimental infections revealed that the bacterial isolates were particularly aggressive in the stems and fruit of pepper and tomato. Biochemical, physiological and serological tests in conjunction with fatty acid profile analysis confirmed that they were Erwinia carotovora subsp. carotovora (Jones) Bergey et al. The product of 434 bp polymerase chain reaction (PCR) enabled a preliminary identification of isolates to be made. Restriction fragment length polymorphism (RFLP) analysis of amplification products showed that the isolates DPP 23_ef and DPP 24_m, strain type CFBP 2046 and DPP 28₁, isolated from pepper fruit, belonged to the RFLP group 12, whereas DPP 29, also isolated from pepper fruit, was included in RFLP group 1. Measures to prevent and control this recently introduced disease are suggested in the conclusion of this paper.     

Behavior of bacteriophage P1 inErwinia carotovora subsp.carotovora

Bacteriophage P1KMclr100 was tranferred toErwinia carotovora subsp.carotovora. P1 was stably maintained as detected by hybridization and transfer of kanamycin resistance. Lysogens ofE. carotovora failed to produce any viable P1 phage. Although total DNA from P1 lysogens ofE. carotovora hybridized to³²P-labeled P1 probe, we were not able to detect P1 DNA as an extrachromosomal element. Attempts to use bacteriophage P1 as a vector for transposon Tn5 insertion mutagenesis inE. carotovora were not successful. Our results indicate that lytic replication of P1 DNA does not occur in P1 lysogens ofE. carotovora and that P1 DNA is probably integrated into the bacterial chromosome.Journal paper 10085 from the Purdue Agricultural Experiment Research Station.     

Revision of two species of ?Ulocrinus and a new pelecocrinid crinoid from West Timor

Two cladid crinoid species, ?Ulocrinus indicus Wanner, 1924, and ?Ulocrinus conoideus Wanner, 1937, are reinterpreted as cladid crinoids that do not belong to the cromyocrinids. This justifies Wanner's questioning of the generic assignment. ?Ulocrinus indicus has bifascial radial facets and is considered an advanced intermediate dendrocrinid assigned to the incertae superfamiliae, Katerocrinidae n. fam., Katerocrinus indicus n. gen., n. comb. This extends the range of the bifascial dendrocrinids upward into the Permian. ?Ulocrinus conoideus has trifascial radial facets and is assigned to the superfamily Scytalocrinoidea, incertae familiae, Dochmocrinus conoideus n. gen., n. comb. It re-emphasizes the need for revision of the family Scytalocrinidae. An unnamed, poorly preserved cup is described and considered to be a pelecocrinid crinoid, thus adding to the diversity of the West Timor Permian crinoids.     

Freckle disease of banana in Hawaii caused by Phyllostictina musarum (Cke.) Petr

‘Freckle’ (‘black-spot’ disease) of bananas is common on leaves and fruit of Dwarf Cavendish and other varieties in Hawaii, especially after rainy periods. On fruit, symptoms may appear 2–4 weeks after the bunch has opened, and become more severe as maturity is approached. The disease is usually confined to older leaves on affected plants. Freckled tissue contains numerous pycnidia of Phyllostictina musarum and disease was experimentally induced by inoculating leaves and fruit with conidia of this fungus. This appears to be the first record of successful inoculation with P. musarum. Conidia of P. musarum germinate after 3–6 h in a film of water on banana peel, appressoria being formed after 18–30 h. Penetration of the epidermis occurs 24–96 h after inoculation, and is brought about by an infection hypha which grows from the appressorium. The progressive increase in severity of freckle as fruit matures is due to repeated infection by further conidia of P. musarum, rather than to enlargement of original infections. Some banana clones, including Gros Michel, appear to be resistant to the fungus Dispersal of P. musarum conidia immediately after discharge from the pycnidium is chiefly by rainwater and dew. Secondary infections contribute greatly to the total number of infections. Conidium dispersal by water often results in the development of characteristic patterns of spotting, chiefly in the form of streaks or circular areas, coinciding with the directions of movement of rainwater and dew. Large numbers of conidia of P. musarum are washed on to fruit in rainwater and dew running from diseased, overhead leaves.     

ATP-induced Changes in Energy Status and Membrane Integrity of Harvested Litchi Fruit and its Relation to Pathogen Resistance

Litchi is a subtropical fruit of high commercial value on the international market but the fruit deteriorates rapidly after harvest due to rot development caused by Peronophythora litchii. To investigate the role of energy metabolism during disease development on harvested litchi fruit, fruits were dipped into solutions of either 0 or 1.0 mm adenosine triphosphate (ATP) for 3 min before being inoculated with Peronophythora litchii or not. Fruit were then stored for 6 days at 25°C and 90–100% relative humidity. Significant reductions in pericarp browning and disease severity and significant delays in membrane permeability and malondialdehyde (MDA) content were found in ATP‐treated and P. litchii‐inoculated fruit. Higher ATP concentrations and adenylate energy charge (EC) were observed in ATP‐treated fruit. In addition, lower activities of phospholipase D, acid phosphatase and lipoxygenase enzymes involved in membrane lipid peroxidation and hydrolysis were recorded in ATP‐treated fruit. Thus, treatment with ATP maintained higher energy levels, inhibited activities of the membrane hydrolysis‐related enzymes, reduced membrane lipid peroxidation and helped maintain membrane integrity of the harvested litchi fruit at the early stage of storage, which could account for the inhibition of disease development of P. litchii‐inoculated fruit.     

Control of green mould of citrus by using Trichoderma harzianum,humic acid or garlic

Penicillium digitatum, an aggressive fungus causes post-harvest decay of mandarin sweet orange and Washington navel. In vitro Trichoderma harzianum or humic acid (HA) or powdered cloves of garlic caused inhibition of fungal growth of isolates P₁ and P₂. Under storage conditions, the fruit citrus is protected by using T. harzianum with standard volume 2.0?ml (9.6?×?10⁶?conidia/ml) and application 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum spore suspension (1.0?×?10⁶?spores/ml) compared to control. Spraying the fruit citrus by standard volume of 2.0?ml of either HA or powder cloves of garlic 1% on each fruit 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum (1.0?×?10⁶ spores/ml) compared to control. The lowest percentage of disease incidence and disease severity were associated with powder of cloves garlic and followed by HA and T. harzianum during two growing seasons compared with the untreated and control.     

Gamma irradiation‐induced disease resistance of pear (Pyrus pyrifolia “Niitaka”) against Penicillium expansum

In this study, the effects of gamma irradiation on the resistance of pear fruit against Penicillium expansum, the causal agent of blue mould disease, were investigated. A low dose of gamma irradiation for 14 days increased the disease resistance and firmness of pear fruits. Remarkably, exposure to 200 Gy of gamma irradiation significantly maintained fruit firmness, markedly reduced disease incidence and enhanced the activity of defence‐related enzymes (e.g., β‐1,3‐glucanase, phenylalanine ammonia lyase, peroxidase and polyphenol oxidase) and expression of pathogenesis‐related (PR) genes (e.g., PR‐1, PR‐3 and PR‐4). Therefore, the gamma irradiation‐induced resistance against P. expansum involves both metabolic changes and the induction of expression of defence‐related genes. In addition, scanning electron microscopic analysis revealed that gamma irradiation significantly inhibits the growth of P. expansum. These results suggest that exposure of mature harvested pear fruits to artificial gamma irradiation confers fungal disease resistance; therefore, gamma irradiation represents an important strategy for controlling postharvest diseases in pear fruit.     

Studies on Pantoea citrea, the Causal Agent of Pink Disease of Pineapple

The causal agent of pink disease of pineapple has been identified as Pantoea citrea, a member of the Enterobacteriaceae. Comparative physiological and biochemical analyses demonstrated that P. citrea isolated from diseased pineapple fruit in the Philippines possesses features identical to those of an American Type Culture Collection type strain of P. citrea and not to those of P. ananas, P. herbicola (formerly Erwinia herbicola), and P. stewartii (formerly Erwinia stewartii). P. citrea induces the production of compounds in pineapple which become pink to reddish-brown upon cooking the fruit, pulp, or juice. This distinct colour is not induced by Escherichia coli, Agrobacterium tumefaciens, Burkholderia gladioli, Pseudomonas fluorescens, Gluconobacter oxydans, Acetobacter aceti, and Acinetobacter calcoaceticus. Like other well characterized bacteria pathogens, such as Pseudomonas syringae pv. phaseolicola, P. citrea elicits the hypersensitive response (HR) in tobacco. By contrast, G. oxydans and A. aceti that have been previously implicated as the causal agents of pink disease, do not elicit HR. Although the nature of the pink colour in pineapple produced by P. citrea has not been elucidated, the locus conferring this activity has been located on its chromosome. The pink colour can be restored in an avirulent, pink colour defective mutant strain, CMC6, by complementation in trans with a specific 3.8 kb genomic DNA fragment of P. citrea. This suggests that P. citrea contains the genetic elements that are required for pink disease.     

Galactose metabolism inErwinia carotovora Subsp.carotovora

The pyrimidine analogue 5-fluorouracil was shown to be a potent inhibitor of the growth ofMethanobacterium thermoautotrophicum strain Marburg (50% inhibition of growth at 1 g ml^–1). The nucleoside, 5-fluorodeoxyuridine, also inhibited growth, but the nucleotide 5-fluorodeoxyuridylate did not inhibit, nor did 5-fluorocytosine. Several nucleobases and nucleosides were used as potential antagonists of fluorouracil and fluorodeoxyuridine. Of these, only uracil in excess over fluorouracil relieved the inhibition of growth. These results imply that a pyrimidine salvage pathway is present inM. thermoautotrophicum. 5-Fluorouracil does not inhibit methane production. Although treated cultures produced less methane than did controls, more than twice as much methane was synthesized per cell. This result suggests that methanogenesis is uncoupled from growth by 5-fluorouracil.     

Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora

Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram-negative bacteria. We have used a λ-sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon-based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein Is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA-induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.     

The Occurrence of Bacterial Red Streak of Sugarcane Caused by Pseudomonas syringae pv. syringae in Iran

A bacterial leaf streak disease characterized by reddish, narrow (1–2 mm wide) streaks of variable size, and occasionally with bleached centers, was found in sugarcane (Saccharum, interspecific hybrid) fields in northern Iran. The incitant bacterium was identified as Pseudomonas syringae pv. syringae (P. s. syringae). The disease is similar in aetiology to the sugarcane ‘red streak’ disease reported recently from Japan. Cultivardependent variations in symptoms were noted., Difference in pathogenicity as well as in electrophoretic profile of cell proteins between strains of P.s. syringae causing red streak in sugarcane and those causing canker on stone fruit trees, were observed.     

Phenotypic and Genetic Diversity of Erwinia carotovora ssp. carotovora Strains from Asia

A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR‐restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S‐23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α‐methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia.     

The Phytophthora diseases of stone fruit trees in Greece

Phytophthora collar and crown rots are serious soilborne diseases which for a long time have caused considerable losses in stone fruit orchards in Greece. A number of Phytophthora species are notorious for being the cause of crown and root rots in Greek stone trees, including P. cactorum, P. citricola, P. cryptogea, P. drechsleri, P. nicotianae, P. citrophthora, P. syringae and P. megasperma. The most important Phytophthora species is P. cactorum, while P. syringae and P. citrophthora may be locally significant. The economic consequences from death of peach trees and yield losses caused by this disease in Imathia County are serious.     

The extent and survival of contamination of potato stocks in Scotland by Erwinia carotovora var. carotovora and E. carotovora var.atroseptica*

The following results were obtained when fifty-seven bulk and crate-stored commercial seed potato stocks from the East of Scotland were examined in 1966-8 for contamination by pectolytic Erwinia spp. (1) Most tubers of all the cultivars and stocks examined, irrespective of whether they were obtained from blackleg-infected or blackleg-free crops, were contaminated with E. carotovora; (2) some 80% of the Erwinia isolates obtained were identified as var. atroseptica, the rest being var. carotovora; (3) the organisms survived in and on tubers for 6–7 months of bulk storage over the winter and up to planting time the following spring; (4) contrary to what is generally thought, the high incidence of contamination of all stocks, while suggesting that the seed itself is the major source of E. carotovora for the growing crop, emphasizes that other factors affect manifestation of blackleg in the field and soft rot in store.     

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

Dispersal of Acalitus essigi to blackberry (Rubus fruticosus agg.) fruit

The eriophyoid mite, Acalitus essigi (Hassan), is hypothesised to be responsible for redberry disease of blackberry (Rubus fruticosus L. agg.) fruit. Infested fruit ripen unevenly with affected drupelets becoming hard, inedible and bright red, whereas unaffected drupelets ripen evenly. As a first step toward possible control of this disease, the method and timing of dispersal of A. essigi onto developing blackberry fruit was examined. No mites were found on unopened flower buds or open flowers. However, infestation of fruit was found to commence during the green stage of fruit development and significantly increase during the red fruit stage. Although redberry symptoms were not observed, experimental exclusion of A. essigi to prevent the mite moving up the pedicel of open flowers by a sticky barrier significantly reduced A. essigi populations within the resultant fruit by over five fold that of control fruit. Although very low levels of aerial dispersal onto fruit cannot be discounted it was concluded that non-aerial or crawling dispersal via the fruit pedicel was the dominant method of blackberry fruit infestation.     

Effect of Chelating Agents on Exopolygalacturonate Lyase of Erwinia carotovora subsp. carotovora

Erwinia exopolygalacturonate lyase is strongly activated by Na⁺, but very weakly by divalent cations such as Ca²⁺, in contrast to most of the known pectic lyases; nevertheless, this enzyme is completely inhibited by 1 mM EDTA. In this work, six polyamino carboxylates such as EDTA and a polyamine were examined for their effects on the enzyme activity.

EDTA, trans-1,2-cyclohexanediaminetetraacetate, and diethylenetriaminepentaacetate inhibited the enzyme strongly, but ethylenediaminediacetate showed little inhibition. Only tri-ethylenetetramine stimulated the enzyme. The removal of EDTA from the enzyme solution resulted in an almost complete restoration of the activity. The EDTA-treated enzyme as well as the untreated one revealed no requirement for any divalent cations. The inactivation by hydroxyethylenediaminetriacetate, a mild inhibitor, was the mixed type. It seems most likely that the inactivation by the polyamino carboxylates is caused not by sequestering any metals but by directly forming an enzyme–chelator complex.     

Biological Control of Green Mould Decay in Postharvest Chinese Bayberries by Pichia membranaefaciens

The effect of the yeast antagonist Pichia membranaefaciens for control of green mould decay caused by Penicillium citrinum or Verticicladiella abietina and natural decay in postharvest Chinese bayberries (Myrica rubra Seib & Zucc.), and the possible mechanisms were investigated. The results showed that 1 × 10⁹ colony‐forming units (CFU)/ml of washed cell suspensions of the yeast provided better control of green mould decay than yeast in culture broth at the same concentration. Treatment with cell‐free culture filtrates or autoclaved cell cultures had little effect on disease incidence. The concentration of a washed cell suspension of P. membranaefaciens had a significant effect on efficacy in controlling disease incidence. At a concentration range from 1 × 10⁶ to 1 × 10⁹ CFU/ml, the higher the concentration of the antagonist, the lower was the incidence of the disease. In the inoculated wounds of Chinese bayberries, populations of P. membranaefaciens increased by approximately 145‐ and 41‐fold, respectively, after incubation at 20°C for 2 day or at 1°C for 8 day. P. membranaefaciens significantly induced activities of two defence‐related enzymes chitinase and β‐1, 3‐glucanase in Chinese bayberries. The in vitro experiment showed that spore germination and germ tube elongation of the two pathogens were markedly inhibited by washed cell suspensions of P. membranaefaciens. In addition, P. membranaefaciens significantly reduced natural decay in Chinese bayberries. These results indicate that P. membranaefaciens can effectively reduce fruit decay possibly by directly inhibiting pathogen growth and indirectly by inducing disease resistance. Thus, we suggest that P. membranaefaciens has potential as a biocontrol agent to control fruit decay in Chinese bayberries during postharvest storage.