Enrique G. Medrano

Involvement of a Plasmid-Encoded Type IV Secretion System in the Plant Tissue Watersoaking Phenotype of Burkholderia cenocepacia

Burkholderia cenocepacia strain K56-2, a representative of the Burkholderia cepacia complex, is part of the epidemic and clinically problematic ET12 lineage. The strain produced plant tissue watersoaking (ptw) on onion tissue, which is a plant disease-associated trait. Using plasposon mutagenesis, mutants in the ptw phenotype were generated. The translated sequence of a disrupted gene (ptwD4) from a ptw-negative mutant showed homology to VirD4-like proteins. Analysis of the region proximal to the transfer gene homolog identified a gene cluster located on the 92-kb resident plasmid that showed homology to type IV secretion systems. The role of ptwD4, ptwC, ptwB4, and ptwB10 in the expression of ptw activity was determined by conducting site-directed mutagenesis. The ptw phenotype was not expressed by K56-2 derivatives with a disruption in ptwD4, ptwB4, or ptwB10 but was observed in a derivative with a disruption in ptwC. Complementation of ptw-negative K56-2 derivatives in trans resulted in complete restoration of the ptw phenotype. In addition, analysis of culture supernatants revealed that the putative ptw effector(s) was a secreted, heat-stable protein(s) that caused plasmolysis of plant protoplasts. A second chromosomally encoded type IV secretion system with complete homology to the VirB-VirD system was identified in K56-2. Site-directed mutagenesis of key secretory genes in the VirB-VirD system did not affect expression of the ptw phenotype. Our findings indicate that in strain K56-2, the plasmid-encoded Ptw type IV secretion system is responsible for the secretion of a plant cytotoxic protein(s).

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.

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).

Temporal analysis of cotton boll symptoms resulting from southern green stink bug feeding and transmission of a bacterial pathogen.

ABSTRACT The southern green stink bug, Nezara viridula (L.), is a significant pest of cotton, Gossypium hirsutum L., and is becoming an increasing challenge due to the decrease in use of broad-spectrum insecticides on the crop. The southern green stink bug can vector an opportunistic Pantoea agglomerans strain (designated Sc 1-R) into cotton bolls, resulting in infection. The appearance of stink bug damage varies, and pest managers cannot readily identify its source. This research reports a systematic depiction of green, immature boll responses at various stages of maturity (1, 2, and 3 wk post-anthesis [WPA]) to stink bug injury and to infection by the vectored cotton pathogen by demonstrating the progression of effects 1, 2, and 3 wk after exposure (WAE). When laboratory-reared adult southern green stink bug not harboring Sc 1-R deposited bacteria into greenhouse-grown bolls at 1, 2, or 3 WPA during feeding/probing, bacteria reached concentrations of 109, 109, and 103 colony-forming units (CFUs)/g tissue, respectively, at 3 WAE, yet caused minimal seed and lint damage regardless of the age of the bolls that were penetrated. Bolls at a maturity of 1 or 2 WPA showed similar susceptibility when exposed to stink bugs that vectored Sc 1-R. After a week of infection, seeds were salmon-pink with normal white lint and up to 104 CFUs/g tissue when Sc 1-R was detected. Necrosis of the entire inoculated locule(s) with a maximum Sc 1-R concentration detected at 108 CFUs/g tissue occurred in samples harvested 2 or 3 WAE. Conversely, seed and lint deterioration due to the transmitted opportunist into bolls exposed 3 WPA was confined to the puncture site. In summary, after a week of development, bolls were tolerant to southern green stink bug feeding/ probing damage and to nonpathogenic bacteria, but they were severely damaged when the opportunistic pathogen Sc 1-R was transmitted. At 3 WPA, the fruit was immune to the spread of the pathogen with infections confined to the puncture site.

Southern Green Stink Bugs (Hemiptera: Pentatomidae) as Vectors of Pathogens Affecting Cotton Bolls — A Brief Review

Abstract. The southern green stink bug, Nezara viridule L., is a member of the stink bug complex that has become more economically important in cotton, Gossypium hirsutum L., production in recent years. A disease of cotton bolls, identified as South Carolina boll rot, seemed coincidental to the increase in abundance of stink bugs. The relationship between the occurrence of the cotton disease and the southern green stink bug is discussed here. This review documents: the identification and description of the disease; potential causal agents and insect vectors; conclusive identification of Pentoea egglomerans (Ewing and Fife) Gavini et al. as the causative agent of the disease; establishment of southern green stink bug as a model insect based on ingestion, retention, and transmission of the disease to cotton bolls; determination of vulnerability of bolls of different ages to disease; and, transmission of other disease agents by southern green stink bugs. Future research needs also are discussed.

Genome Sequence of Pantoea sp. Strain Sc 1, an Opportunistic Cotton Pathogen

Pantoea is comprised of a broad spectrum of species, including plant pathogens. Here, we provide an annotated genome sequence of Pantoea sp. strain Sc 1, which was isolated from a diseased cotton boll. This research provides the first genome sequence of a bona fide Pantoea sp. insect-vectored cotton pathogen.

Analysis of Microscopic Injuries Caused by Southern Green Stink Bug (Hemiptera: Pentatomidae) Feeding on Cotton Bolls

Abstract. The southern green stink bug, Nezara viridula (L.), utilizes stylets while feeding to pierce the wall of a boll of cotton, Gossypium hirsutum L., and can inoculate disease-causing pathogens into developing green bolls. Detection of diseased bolls is difficult because the surface of the carpel wall frequently lacks apparent macroscopic evidence of insect feeding regardless of infection; blisters on the interior surface of the carpel wall are commonly masked by darkened necrotic tissue. The objectives of this study were to characterize microscopic evidence of feeding by laboratory-reared southern green stink bugs on greenhouse-grown cotton bolls and to use these findings to aid in detection of feeding evidence by piercing-sucking insects on field-collected bolls from four southeastern states. Microscopic analyses of greenhouse-reared bolls fed upon by southern green stink bug (n = 40) yielded definitive characteristics and imagery of feeding evidence. Salivary flanges on the exterior surface of the boll and blisters at the feeding site on the interior surface of the carpel wall were characteristic of feeding by southern green stink bug. Also, salivary flanges were associated with all external puncture wounds on the boll wall. These characteristics were used as the baseline to detect feeding by feral piercing-sucking pests on field-grown bolls during the 2008 and 2009 production seasons. Injury characteristics from field-grown bolls were comparable to characteristics observed in greenhouse-reared bolls after feeding by southern green stink bug. A few field-collected bolls (5%; n = 160) possessed discolored seeds and exhibited evidence of boll feeding externally but lacked blisters on the interior surface of the carpel wall, thus suggesting an incomplete breach of the interior carpel wall. Callus tissue, colloquially termed a ‘wart,’ on the interior surface of the boll carpel wall is typically associated with feeding by stink bugs but was not detected in bolls grown in a greenhouse and was only sporadically detected in bolls collected from the field. When present, callus tissue was always associated with an external puncture. This is the first study to unequivocally illustrate microscopic characteristics associated with feeding damage by southern green stink bug on cotton bolls. Findings are discussed in relation to evidence of feeding by insects in field-collected bolls of unknown age and history.

Complete Genome Sequence of a Klebsiella pneumoniae Strain Isolated from a Known Cotton Insect Boll Vector

ABSTRACT Klebsiella pneumoniae (associated with bacterial pneumonia) was previously isolated from Nezara viridula, a significant vector of cotton boll-rot pathogens. We provide the first annotated genome sequence of the cotton opportunistic strain K. pneumoniae 5-1. This data provides guidance to study the bases of cotton pathogenesis by bacteria associated with vectors.

Genome Sequence of Pantoea ananatis Strain CFH 7-1, Which Is Associated with a Vector-Borne Cotton Fruit Disease

ABSTRACT Pantoea ananatis is a bacterium with versatile niches that vary from pathogenic to beneficial. We present the genome of strain CFH 7-1, which was recovered from a diseased greenhouse cotton boll previously caged with a field-collected cotton fleahopper (Pseudatomoscelis seriatus). These data will assist in deciphering the infection process.

Relationship Between Piercing-Sucking Insect Control and Internal Lint and Seed Rot in Southeastern Cotton (Gossypium hirsutum L.)

ABSTRACT In 1999, crop consultants scouting for stink bugs (Hemiptera spp.) in South Carolina discovered a formerly unobserved seed rot of cotton that caused yield losses ranging from 10 to 15% in certain fields. The disease has subsequently been reported in fields throughout the southeastern Cotton Belt. Externally, diseased bolls appeared undamaged; internally, green fruit contain pink to dark brown, damp, deformed lint, and necrotic seeds. In greenhouse experiments, we demonstrated transmission of the opportunistic bacterium Pantoea agglomerans by the southern green stink bug, Nezara viridula (L.). Here, green bolls were sampled from stink bug management plots (insecticide protected or nontreated) from four South Atlantic coast states (North Carolina, South Carolina, Georgia, and Florida) to determine disease incidence in the field and its association with piercing—sucking insects feeding. A logistic regression analysis of the boll damage data revealed that disease was 24 times more likely to occur (P = 0.004) in bolls collected from plots in Florida, where evidence of pest pressure was highest, than in bolls harvested in NC with the lowest detected insect pressure. Fruit from plots treated with insecticide, a treatment which reduced transmission agent numbers, were 4 times less likely to be diseased than bolls from unprotected sites (P = 0.002). Overall, punctured bolls were 125 times more likely to also have disease symptoms than nonpunctured bolls, irrespective of whether or not plots were protected with insecticides (P = 0.0001). Much of the damage to cotton bolls that is commonly attributed to stink bug feeding is likely the resulting effect of vectored pathogens.