Cotton (Gossypium hirsutum L.) boll rotting bacteria vectored by the brown stink bug, Euschistus servus (Say) (Hemiptera: Pentatomidae).

AIM: To determine the capacity of the brown stink bug (BSB) (Euschistus servus) to transmit an infective Pantoea agglomerans strain Sc 1-R into cotton (Gossypium hirsutum) bolls. METHODS AND RESULTS: A laboratory colony of BSB was maintained on fresh green beans. Either sterile or Sc 1-R contaminated beans were offered to adult insects. Strain Sc 1-R holds rifampicin resistance (Rif(r) ). Insects were then caged with unopened greenhouse-grown bolls. After 2 days, BSB were surface sterilized, ground, and then plated on media with and without Rif. Two weeks later, seed with lint were ground and plated on media with and without Rif. Microbes were recovered on nonselective media from all BSBs and from seed/lint at concentrations reaching 10(9)  CFU g(-1) tissue. Rif(r) bacteria were recovered strictly from insects exposed to Sc1-R and from diseased seed/lint of respective bolls. CONCLUSIONS: Euschistus servus was capable of transmitting strain Sc 1-R into bolls resulting in disease. Insects not exposed to the pathogen deposited bacteria yet the nonpathogenic microbes produced insignificant damage to the boll tissue. SIGNIFICANCE AND IMPACT OF STUDY: This is the first study to concretely show the capacity of the BSB to transmit an infective P. agglomerans strain resulting in boll disease.

Phytotoxicity of fusaric acid and analogs to cotton.

We developed a cotton cotyledonary leaf bioassay to test the phytotoxicity of fusaric acid (5-butylpicolinic acid), picolinic acid and related analogs. The compounds were dissolved in aqueous Tween 80, and 20 µL of the test solution was placed at three positions on the leaf, and a needle was used to puncture the leaf through each drop; the results were evaluated after 48 h. In contrast to previous studies, we found the carboxylic acid group is essential for phytotoxicity. Nicotinic acid was considerably less phytotoxic than picolinic acid and conversion of picolinic acid to the amide or N-oxide decreased phytotoxicity. Increasing the alkyl chain length at the 5-position on picolinic acid from two up to five carbons atoms increased phytotoxicity. Fusaric acid methyl ester, the most phytotoxic compound tested, is a naturally occurring compound; as such it has potential as a herbicide in organic farming.

Transmission of cotton seed and boll rotting bacteria by the southern green stink bug (Nezara viridula L.).

AIMS: To determine the ability of the southern green stink bug (SGSB) (Nezara viridula L.) to transmit Pantoea agglomerans into cotton (Gossypium hirsutum) bolls. METHODS AND RESULTS: An SGSB laboratory colony was kept on fresh green beans. A P. agglomerans variant resistant to rifampicin (Rif) (strain Sc 1-R) was used as the opportunistic cotton pathogen. Adult insects were individually provided green beans that were sterilized and then soaked in either sterile water or in a suspension of strain Sc 1-R. Insects were individually caged with an unopened greenhouse-grown cotton boll. After 2 days, live SGSB were collected, surfaced sterilized, ground, serially diluted, and then plated on nonselective media and media amended with Rif. Exterior and interior evidence of feeding on bolls was recorded 2 weeks after exposure to insects. Seed and lint tissue were harvested, ground, serially diluted, and then plated on media with and without Rif. Bacteria were recovered on nonselective media from all insects, and from seed and lint with signs of insect feeding at concentrations ranging from 10(2) to 10(9) CFU g(-1) tissue. The Sc 1-R strain was isolated only from insects exposed to the marked strain and from seed and lint of respective bolls showing signs of insect feeding. Evidence of insect feeding on the exterior wall of the carpel was not always apparent (47%), whereas feeding was always observed (100%) on the interior wall in association with bacterial infections of seed and lint. CONCLUSIONS: Nezara viridula readily ingested the opportunistic P. agglomerans strain Sc 1-R and transmitted it into unopened cotton bolls. Infections by the transmitted Sc 1-R strain caused rotting of the entire locule that masked internal carpel wounds incurred by insect feeding. Bacteria were recovered from penetration points by insects not exposed to the pathogen, but locule damage was limited to the area surrounding the feeding site (c. 3 mm). SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that demonstrates the ability of SGSB to acquire and transmit plant pathogenic bacteria into cotton bolls.

Role of Pantoea agglomerans in opportunistic bacterial seed and boll rot of cotton (Gossypium hirsutum) grown in the field.

AIMS: To investigate the aetiology of seed and boll rot of cotton grown in South Carolina (SC). METHODS AND RESULTS: Bacteria were isolated from diseased locules of cotton bolls collected in a field in SC, USA and tested for the ability to cause comparable disease symptoms in greenhouse grown cotton fruit. Spontaneously generated rifampicin-resistant (Rif(r)) mutants of the isolates were used in confirmatory pathogenicity tests. Resistance to the antibiotic was both stable and effective in differentiating between an inoculated Rif(r) strain, rifampicin-sensitive contaminants and/or endophytes. A series of inoculation methods was tested at various boll developmental stages and at different fruiting nodes on the plant. Field disease symptoms were reproduced by inoculating bolls at 2 weeks postanthesis with bacterial suspensions ranging from 10(3) to 10(6) CFU ml(-1). Pathogenic isolates were categorized as Pantoea agglomerans on the basis of phenotype testing, fatty acid profiling (similarity index = 0.94), and 16s ribosomal DNA sequence analysis (99% nucleotide identity). CONCLUSIONS: Pantoea agglomerans isolates from field-collected immature, diseased cotton caused comparable infection symptoms in greenhouse produced cotton fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: In 1999, significant yield losses in SC cotton resulted from a previously unobserved seed and boll rot that has since been reported in other southeastern states. This study demonstrated a role of P. agglomerans in producing opportunistic bacterial seed and boll rot of cotton.

Induction of delta-cadinene synthase and sesquiterpenoid phytoalexins in cotton by Verticillium dahliae.

Phytoalexin biosynthesis occurred earlier in the resistant cotton cultivar Seabrook Sea Island 12B2 (SBSI) (Gossypium barbadense) than in the susceptible cotton cultivar Rowden (G. hirsutum) after inoculation with a defoliating isolate of the pathogen Verticillium dahliae. This was demonstrated by significantly higher levels of phytoalexins in SBSI 12 h after inoculation. Furthermore, by 48 h after inoculation of SBSI, the phytoalexins hemigossypol and desoxyhemigossypol achieved levels (23.9 and 10.5 microgram/g of fresh tissue, respectively) sufficient to completely inhibit conidial germination. Rowden required 96 h to attain comparable levels. Similarly, the activity of delta-cadinene synthase, a key enzyme required for the biosynthesis of the terpenoid phytoalexins, increased more rapidly in the resistant cotton cultivar than in the susceptible one. The changes in phytoalexin concentrations and enzyme activity are consistent with the hypothesis that phytoalexins are an essential component in protecting the plant from infection by V. dahliae.

Cardiovascular responses produced by microinjection of serotonin-receptor agonists into the paraventricular nucleus in conscious rats.

Activation of serotonin (5-hydroxytryptamine, 5-HT) receptors in the brain produces cardiovascular responses by altering autonomic outflow. The paraventricular nucleus (PVN) contains a modest density of 5-HT receptors and has connections to autonomic centers. Experiments were designed to determine whether cardiovascular responses were produced by the administration of 5-HT2- and 5-HT1A-receptor agonists into the PVN of conscious rats. The microinjection of the 5-HT2-receptor agonist DOI [(+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] into the PVN produced dose-dependent (1-10 nmol) increases in heart rate and blood pressure; the peak responses were +39 +/- 10 beats/min and +6 +/- 2 mm Hg, respectively. Both responses were blocked by the concomitant administration of the selective 5-HT2-receptor antagonist LY53857 into the PVN. By contrast, the microinjection of the selective 5-HT1A-receptor agonist R(+)-8-OH-DPAT [R(+)8-hydroxy-2-(di-n-propylamino) tetralin HBr; 1-10 nmol] into the PVN did not affect blood pressure or heart rate. These data suggest that 5-HT neurons projecting from the raphe nuclei to or near the PVN can participate in the central control of the cardiovascular system by way of 5-HT2 receptors. Apparently 5-HT neurons terminating in the PVN can increase blood pressure and heart rate and produce sympathoadrenal activation, metabolic and hormonal responses consistent with those observed in several different stress paradigms.

Mechanisms of Subsurface Drip Irrigation-Mediated Suppression of Lettuce Drop Caused by Sclerotinia minor.

ABSTRACT Subsurface drip irrigation and associated mandatory minimum tillage practices significantly reduced the incidence of lettuce drop (Sclerotinia minor) and the severity of corky root on lettuce compared with furrow irrigation and conventional tillage. Three possible mechanisms for the drip irrigation-mediated disease suppression were examined in this study: qualitative and quantitative differences in the soil microflora under furrow and subsurface drip irrigation; their antagonism and potential bio-control effects on S. minor; and the physical distribution of soil moisture and temperature relative to the two irrigation methods. To determine if the suppressive effects under subsurface drip irrigation were related to changes in soil microflora, soils were assayed for actinomycetes, bacteria, and fungi during the spring and fall seasons. The effects of the irrigation methods on microbial populations were nearly identical during both seasons. In the spring season, the total number of fungal colonies recovered on potato dextrose agar amended with rose Bengal generally was greater in soils under drip irrigation than under furrow irrigation, but no such differences were observed during the fall. Numbers of actinomycetes and bacteria were not significantly different between irrigation methods during either season. No interaction between sampling time and irrigation methods was observed for any of the microbial populations during both seasons. Thus, the significant effect of sampling time observed for actinomycete and bacterial populations during the spring was most likely not caused by the irrigation treatments. There were also no qualitative differences in the three groups of soil microflora between the irrigation treatments. Even though some fungal, actinomycete, and bacterial isolates suppressed mycelial growth of S. minor in in vitro assays, the isolates came from both subsurface drip- and furrow-irrigated soils. In in planta assays, selected isolates failed to reduce the incidence of drop in lettuce plants. The soil moisture under subsurface drip irrigation was significantly lower at all depths and distances from the bed center after an irrigation event than under furrow irrigation. The soil temperature, in contrast, was significantly higher at both 5 and 15 cm depths under drip irrigation than under furrow irrigation. The suppression of lettuce drop under subsurface drip irrigation compared with furrow irrigation is attributed to differential moisture and temperature effects rather than to changes in the soil microflora or their inhibitory effects on S. minor.

Diversity in cyclic sesquiterpene production by Gossypium hirsutum.

Major sesquiterpene components of oil of Texas Race Stock 810 of Gossypium hirsutum were alpha- and beta-selinene. This is the seventh cyclic terpene type found to date in this genus. Both alpha- and beta-selinene, along with aromadendrene, were found but only as minor components of extracts of several domestic cultivars of G. hirsutum.

Differential induction of 3-hydroxy-3-methylglutaryl CoA reductase in two cotton species following inoculation with Verticillium.

Gossypium barbadense cottons are typically more resistant to wilt pathogens than are Gossypium hirsutum cultivars. Both species make terpenoid phytoalexins in response to infection, implicating isoprenoid biosynthesis as a factor in resistance. Conserved regions in plant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the first enzyme in the terpene biosynthesis pathway, were used to design polymerase chain reaction primers for cloning a fragment of a cotton HMGR gene. The clone was used as a probe on Northern blots to show that induction of HMGR mRNA following introduction of Verticillium dahliae spores into the vascular system is much more rapid in Seabrook Sea Island, a restant G. barbadense cotton, than it is in Rowden, a susceptible G. hirsutum. The amount of HMGR mRNA returned to near control levels in 4 days in the former variety but continued to accumulate in the latter. Specific enzyme activity of HMGR also increased more rapidly in stele extracts of Seabrook Sea Island than in Rowden.

Toxicity of cotton phytoalexins to zoopathogenic fungi.

The sesquiterpenoid phytoalexins desoxyhemigossypol, desoxymethoxyhemigossypol, and hemigossypolone formed in cotton (Gossypium hirsutum and G. barbadense) stem xylem infected with Verticillium dahliae were shown to be highly toxic to zoopathogenic fungi. This appears to be the first study of the toxicity of terpenoid phytoalexins to zoopathogenic fungi. The toxicities of the phytoalexins expressed as MIC (micrograms ml-1) values were 8 to 128 against four isolates of Candida albicans and one isolate of Cryptococcus neoformans. These highly toxic compounds or their derivatives may prove useful for the treatment of animal mycoses.

Accurate and Precise Coulometric Determination of Sulfur Dioxide in Compressed Gas Mixtures.

Coulometry has been established as an important and reliable method for the determination of acidic compounds. The analytical method and simple apparatus described here arc applied to the precise and accurate determination of sulfur dioxide in nitrogen, specifically in compressed gas cylinders at nominal concentrations of 50 and 100 µmol/mol (ppm). This method is constant current coulometry where the magnitude of the current is set by the balance between the electrochemical generation of OH-, the flow of SO2, and the chemical reaction of the solution. The method is direct, rapid, and can be refined further to provide analysis at the nanomol/mol level.

Development of a Coulometric Method for Assessing the Concentration of Ambient Levels of CO2/Air in Compressed Gas Mixtures.

The coulometric method presented here is a reliable method for the direct analysis of CO2/air cylinder gas mixtures. It is based on Faraday's laws of electrolysis and therefore no external standardization is required. A series of CO2/air cylinder gas mixtures ranging in concentration from 300 to 375 µmol/mol (ppm) were analyzed and the results compared to those results obtained by non-dispersive infrared (NDIR) analysis with traceability to gravimetric standards. The coulometric method is rapid, sensitive, precise, and with the proper experimental controls, will yield accurate results.

Histochemical localization of desoxyhemigossypol, a phytoalexin in Verticillium dahliae-infected cotton stems.

The terpenoid phytoalexin desoxyhemigossypol (dHG) was detected histochemically in the stem xyiem of Verticillium dahliae Kleb-infected, wilt-resistant Seabrook Sea Island cotton as a green product on V. dahliae mycelium within vessel lumens and in specialized, often solitary, paravascular parenchyma cells. The SbCl3 -HClO4 histochemical reagent yielded a green-coloured Sb-dHG product specific for dHG when used as a spray on chromatograms of extracts from Verticillium-infected stele tissue. Both dHG and related terpenoid aldehyde derivatives occurred together in parenchyma cells and on V. dahliae mycelium. The presence of dHG on Verticillium mycelium reinforces previous studies that identified dHG as the most toxic and possibly most important phytoalexin in the resistance of cotton to V. dahliae.

Histamine release from mast cells by terpenoid aldehydes isolated from glanded varieties of cotton.

In vivo and in vitro experiments have strongly indicated that mast cell degranulation, with its release of histamine and other pharmacoactive compounds, plays a major role in the acute respiratory response of humans following inhalation of cotton textile dust. Thirteen terpenoid aldehydes isolated from the glands of the two major Gossypium species used for cotton production, stimulated significant release of histamine from mast cells at concentrations of 1 micrograms/mL. Eleven of the thirteen compounds produce significant mast cell degranulation at concentrations well below the levels of free terpenoid aldehydes that could be expected to enter the lungs during an eight hour work day under the current permissible card room standards of 200 micrograms per cubic meter. Daily mast cell degranulation, stimulated by these terpenoid aldehydes could account for many of the pathophysiological changes found in the chronic byssinotic.

Ultrastructural and chemical distinction of melanins formed by Verticillium dahliae from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine.

Microsclerotia of three melanin-deficient mutants of Verticillium dahliae formed malanin from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine. The melanins formed from (+)-scytalone or 1,8-dihydroxynaphthalene resembled wild-type melanin chemically and ultrastructurally, whereas the melanins formed from catechol and L-3,4-dihydroxyphenlalanine were different. This suggests that scytalone and 1,8-dihydroxynaphthalene but no catechol or L-3,4-dihydroxyphenylalanine are natural intermediates of melanin biosynthesis in V. dahliae.