John A. McInroy

Survey of indigenous bacterial endophytes from cotton and sweet corn

The genotypic diversity of indigenous bacterial endophytes within stems and roots of sweet corn (Zea mays L.) and cotton (Gossypium hirsutum L.) was determined in field trials throughout one growing season. Strains were isolated from surface-disinfested tissues and identified by fatty acid analysis. Gram-negative bacteria comprised 70.5% of the endophytic bacteria and 27 of the 36 genera identified. The most frequently isolated groups from sweet corn roots, were Burkholderia pickettii and Enterobacter spp.; from sweet corn stems, Bacillus megaterium. Bacterial genera present in sweet corn roots were also generally present in sweet corn stems. However, Burkholderia gladioli, Burkholderia solanacearum and Enterobacter cloacae were isolated much more frequently from sweet corn roots than stems, whereas Methylobacterium spp. were found more frequently in sweet corn stems than roots. Agrobacterium radiobacter, Serratia spp. and Burkholderia solanacearum, were the most frequently isolated groups from cotton roots; and Bacillus megaterium and Bacillus pumilus from cotton stems. Acinetobacter baumannii and Arthrobacter spp. were present in cotton stems but not in cotton roots. There were 14 taxonomic groups present in cotton roots that were not in cotton stems; all but one were Gram-negative. These included, Agrobacterium radiobacter, Bacillus megaterium, Bacillus pumilus, Enterobacter asburiae, Pseudomonas chlororaphis, Serratia spp. and Staphylococcus spp. Rhizobium japonicum and Variovorax paradoxus were isolated, almost exclusively, from the roots of both crops. Bacterial taxa present in both sweet corn and cotton early in the season were generally present late in the season. The diversity of bacteria was greater in roots than stems for each crop.

Induced systemic protection against tomato late blight elicited by plant growth-promoting rhizobacteria.

ABSTRACT Two strains of plant growth-promoting rhizobacteria (PGPR), Bacillus pumilus SE34 and Pseudomonas fluorescens 89B61, elicited systemic protection against late blight on tomato and reduced disease severity by a level equivalent to systemic acquired resistance induced by Phytophthora infestans or induced local resistance by chemical inducer beta-amino butyric acid (BABA) in greenhouse assays. Germination of sporangia and zoospores of P. infestans on leaf surfaces of tomato plants treated with the two PGPR strains, pathogen, and chemical BABA was significantly reduced compared with the noninduced control. Induced protection elicited by PGPR, pathogen, and BABA were examined to determine the signal transduction pathways in three tomato lines: salicylic acid (SA)-hydroxylase transgenic tomato (nahG), ethylene insensitive mutants (Nr/Nr), and jasmonic acid insensitive mutants (def1). Results suggest that induced protection elicited by both bacilli and pseudomonad PGPR strains was SA-independent but ethylene- and jasmonic acid-dependent, whereas systemic acquired resistance elicited by the pathogen and induced local resistance by BABA were SA-dependent. The lack of colonization of tomato leaves by strain 89B61 suggests that the observed induced systemic resistance (ISR) was due to systemic protection by strain 89B61 and not attributable to a direct interaction between pathogen and biological control agent. Although strain SE34 was detected on tomato leaves, ISR mainly accounted for the systemic protection with this strain.

Rhizosphere bacteria antagonistic to soybean cyst (Heterodera glycines) and root-knot (Meloidogyne incognita) nematodes : identification by fatty acid analysis and frequency of biological control activity

Rhizosphere bacteria were isolated from roots of young and mature plants with known antagonism to phytopathogenic nematodes, including velvet bean (Mucuna deeringiana), castor bean (Ricinus communis), sword bean (Cannavalia ensiformis), and Abruzzi rye (Secale cereale). Isolates from antagonistic plants were compared to soybean isolates for the frequency of antagonism to the root-knot (Meloidogyne incognita) and soybean cyst (Heterodera schachtii) nematodes in a disease assay with soybean. Bacterial isolates were identified using fatty acid analysis, and isolates which exhibited a significant reduction in incidence of soybean damage from both nematodes were characterized physiologically. The bacterial taxa associated with antagonistic plants were markedly different from soybean bacteria. Isolates from soybean were predominantly Bacillus spp., while those from antagonistic plants included more coryneform and Gram-negative genera. Pseudomonas cepacia and Pseudomonas gladioli were predominant among Gram-negative bacteria on antagonistic plants but were not isolated from soybean. Four to six times the number of bacteria from antagonistic plants, compared to soybean, significantly reduced disease incidence of both nematodes. No single pattern of physiological reactions was common among all these bacteria, suggesting that multiple mechanisms accounted for the observed biological control. The results suggest that rhizospheres of antagonistic plants may be useful sources of potential biological control agents for phytopathogenic nematodes.

Analysis of populations and physiological characterization of microorganisms in rhizospheres of plants with antagonistic properties to phytopathogenic nematodes

Populations of rhizosphere microflora of plants which have demonstrated an antagonism toward phytopathogenic nematodes, including velvet bean (Mucuna deeringiana), castor bean (Ricinus communis), sword bean (Cannavalia ensiformis), and Abruzzi rye (Secale cereale)., were compared to the rhizosphere microflora of soybean. Population densities of total bacteria were significantly lower for young Abruzzi rye, mature velvet bean, and mature castor bean, and fungi from mature velvet bean than for soybean. Population densities of spore-forming bacilli were significantly higher for Abruzzi rye than for soybean. Population densities of coryneform bacteria for mature sword bean and velvet bean were significantly higher than for soybean. All seedling test poants supported significantly higher population densities of chitinolytic fungi than soybean. On mature plants, chitinolytic bacteria were significantly higher on all test plants except velvet bean. Populations of endophytic root bacteria for three of the four test plants were significantly higher than for soybean. Fifty randomly, selected bacterial strains from seedlings and mature plants of soybean and each test plant were characterized for various physiological traits associated with rhizosphere competence, including chitinolytic activity, gelatin hydrolysis, production of hydrogen cyanide, starch hydrolysis, phenol oxidation, siderophore production, and production of antifungal compounds (inhibition ofPythium ultimum and/orRhizoctonia solani). There was a strong trend to increased frequency in each of the physiological tests with bacteria from test plants in comparison to those from soybean. The frequency of starch hydrolysis was up to 24 times greater for strains from test plants than for soybean strains, and siderophore production was up to 22 times more frequent for test plants. These results demonstrate that, compared to soybean, plants with properties antagonistic to phytopathogenic nematodes have a distinct rhizosphere microflora.

Development of Delivery Systems for Introducing Endophytic Bacteria into Cotton

Experiments were designed to evaluate the effectiveness of several methods for delivering 15 endophytic bacteria into cotton stem and root tissues. The delivery methods included stab-inoculation of bacteria into stems, soaking seeds in bacterial suspensions, methyl cellulose seed coating, foliar spray, bacteria-impregnated granules applied in-furrow, vacuum infiltration and pruned-root dip. The success of delivery was gaged by recovery of the bacteria from internal plant tissues 2 weeks after the plants had been grown in a glasshouse potting mix. Following stab-inoculation into stems or radicles, 10 of the bacterial endophytes which previously exhibited biological control against fusarium wilt of cotton were successfully re-isolated from 50% of the plants inoculated; however, this method was labor-intensive, involved wounding the plant and sometimes reduced plant growth. Four of the other methods established from six to eight of the 15 strains, and, with some strains, all methods effectively established e...

Comparative identification by fatty acid analysis of soil, rhizosphere, and geocarposphere bacteria of peanut (Arachis hypogaea L.)

Bacterial isolates were collected from the geocarposphere, rhizosphere, and root-free soil of field grown peanut (Arachis hypogaea L.) at three sample dates, and the isolates were identified by analysis of fatty acid methyl-esters to determine if qualitative differences exist among the bacterial microflora of these zones. Five bacterial genera were associated with isolates from soil, while pod and root isolates constituted 16 and 13 genera, respectively, indicating that bacterial diversity was higher in the rhizosphere and geocarposphere than in soil. The dominant (most frequently identified) genus across all three samples dates was Flavobacterium, for pods, Pseudomonas for roots, and Bacillus, for root-free soil. Sixteen bacterial taxa were only isolated from the geocarposphere, 7 only from the rhizosphere, and 5 only from soil. These results show that specific bacterial taxa are preferentially adapted to colonization of the geocarposphere and suggest that the soil, rhizosphere, and geocarposphere constitute three distinct ecological niches. Bacteria which colonize the geocarposphere should be examined as potential biological control agents for pod-invading fungi such as the toxigenic strains of Aspergillus flavus and A. parasiticus.

A Pathogenic Fungi Diphenyl Ether Phytotoxin Targets Plant Enoyl (Acyl Carrier Protein) Reductase

Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. We report that this natural diphenyl ether inhibits Arabidopsis (Arabidopsis thaliana) enoyl (acyl carrier protein) reductase (ENR). This enzyme is also sensitive to triclosan, a synthetic antimicrobial diphenyl ether. Whereas cyperin was much less potent than triclosan on this target site, their ability to cause light-independent disruption of membrane integrity and inhibition of ENR is similar at their respective phytotoxic concentrations. The sequence of ENR is highly conserved within higher plants and a homology model of Arabidopsis ENR was derived from the crystal structure of the protein from Brassica napus. Cyperin mimicked the binding of triclosan in the binding pocket of ENR. Both molecules were stabilized by the π-π stacking interaction between one of their phenyl rings and the nicotinamide ring of the NAD+. Furthermore, the side chain of tyrosine is involved in hydrogen bonding with a phenolic hydroxy group of cyperin. Therefore, cyperin may contribute to the virulence of the pathogens by inhibiting ENR and destabilizing the membrane integrity of the cells surrounding the point of infection.

Masking of antibiotic-resistance upon recovery of endophytic bacteria

During studies on internal plant colonization by rhizosphere bacteria and endophytic bacteria over several years, we frequently observed lack of growth of rifampicin-resistant mutants (rif+) on tryptic soy agar amended with rifampicin (RTSA). Following seed treatment of cucumber with 6 species of rif+ rhizosphere bacteria in one experiment, all strains were recoverable on RTSA when external root colonization was monitored. Following trituration of surface-disinfested roots, only one strain grew directly on RTSA; however colonies isolated on tryptic soy agar (TSA) grew within 18 h after transfer to RTSA. We term this temporary loss of the antibiotic-resistant phenotype ‘antibiotic masking’. Antibiotic masking was also observed with isolation of 7 rif+ endophytic bacterial strains from inside stems of cotton and with isolation of mutants of bacterial endophytes resistant to polymyxin B sulfate from cotton plants. Rifampicin-masking was not accounted for in vitro by inhibitory compounds from cotton plant extracts, by bacterial growth on low nutrient agar, or by competition with other bacteria. Collectively, these results suggest that expression of antibiotic-resistance may be altered in planta, although causes for this antibiotic-masking remain to be elucidated, methods for quantifying internal plant colonization by rif+ bacteria should account for this possibility. ei]Section editor: R O D Dixon

Elizabethkingia endophytica sp. nov., isolated from Zea mays and emended description of Elizabethkingia anophelisKämpfer et al. 2011.

A slightly yellow bacterial strain (JM-87(T)), isolated from the stem of healthy 10 day-old sweet corn (Zea mays), was studied for its taxonomic allocation. The isolate revealed Gram-stain-negative, rod-shaped cells. A comparison of the 16S rRNA gene sequence of the isolate showed 99.1, 97.8, and 97.4% similarity to the 16S rRNA gene sequences of the type strains of Elizabethkingia anophelis, Elizabethkingia meningoseptica and Elizabethkingia miricola, respectively. The fatty acid profile of strain JM-87(T) consisted mainly of the major fatty acids C15:0 iso, C17:0 iso 3-OH, and C15:0 iso 2-OH/C16:1ω7c/t. The quinone system of strain JM-87(T) contained, exclusively, menaquinone MK-6. The major polyamine was sym-homospermidine. The polar lipid profile consisted of the major lipid phosphatidylethanolamine plus several unidentified aminolipids and other unidentified lipids. DNA-DNA hybridization experiments with E. meningoseptica CCUG 214(T) ( = ATCC 13253(T)), E. miricola KCTC 12492(T) ( = GTC 862(T)) and E. anophelis R26(T) resulted in relatedness values of 17% (reciprocal 16%), 30% (reciprocal 19%), and 51% (reciprocal 54%), respectively. These DNA-DNA hybridization results, in addition to some differentiating biochemical properties, clearly indicate that strain JM-87(T) is a representative of a novel species, for which the name Elizabethkingia endophytica sp. nov. is proposed. The type strain is JM-87(T) ( = CIP 110885(T) = LMG 28604(T) = CCM 8570(T)).

Chryseobacterium gallinarum sp. nov., isolated from a chicken, and Chryseobacterium contaminans sp. nov., isolated as a contaminant from a rhizosphere sample

Two yellow-pigmented bacterial strains (100(T) and C26(T)), showing 98.4 % 16S rRNA gene sequence similarity to each other and isolated from a chicken in Germany and as a contaminant from an agar plate of a rhizosphere sample in Alabama, were studied by using a polyphasic taxonomic approach. Cells of both isolates were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequences of the two organisms with the sequences of the type strains of the most closely related species of the genus Chryseobacterium showed the highest sequence similarities of strains 100(T) and C26(T) to the type strains of Chryseobacterium joostei (respectively 97.5 and 98.2 %), C. viscerum (96.6, 97.8 %), C. gleum (97.1, 97.7 %), C. arthrosphaerae (97.3%, 97.7 %), C. indologenes (97.2, 97.7 %), C. tructae (96.6, 97.6 %), C. jejuense (97.0, 97.6 %) and C. oncorhynchi (96.3, 97.5 %); 16S rRNA gene sequence similarities to members of all other species of the genus Chryseobacterium were below 97.5 %. The fatty acid profiles of both strains consisted of the major fatty acids iso-C15 : 0, summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C17 : 1ω9c and iso-C17 : 0 3-OH, but also showed slight differences (absence or presence of C16 : 0 3-OH and iso-C15 : 1 F). DNA-DNA hybridizations between the two strains and between the novel strains and the type strains of C. joostei, C. indologenes, C. jejuense, C. tructae and C. viscerum resulted in relatedness values clearly below 70 %. These DNA-DNA hybridization results and the differentiating biochemical and chemotaxonomic properties showed that both strains 100(T) and C26(T) represent novel species, for which the names Chryseobacterium gallinarum sp. nov. (type strain 100(T) = LMG 27808(T) = CCM 8493(T)) and Chryseobacterium contaminans sp. nov. (type strain C26(T) = LMG 27810(T) = CCM 8492(T)) are proposed.