Demonstration that a Klebsiella pneumoniae subsp. pneumoniae isolated from an insect (Nezara viridula) harbors a plasmid-borne type IV secretion system.

Previously, we reported the isolation of Klebsiella pneumoniae subspecies pneumoniae strain Kp 5-1 from a southern green stink bug (Nezara viridula) that is a significant pest of numerous economically important crops. We subsequently sequenced the strains whole genome. Here, we report the presence of a functional plasmid-borne type IV secretion (TFSS) system that was identified using genomic mining of the annotated genome. Comparison of the Kp 5-1 resident 186 kb plasmid (pKp 5-1) with nine other Klebsiella with plasmids of comparable size from clinical and environmental strains revealed putative TFSS with identities ranging from 70 to 99%. A primer set was designed at the pKp 5-1 region that shared homology with traC of the conjugation capable F-plasmid. The 2.4 kb amplified PCR product was cloned, sequenced, and used in hybridization experiments verify that the predicted gene was extra-chromosomally located. Based on biparental mating experimental results, a K. pneumoniae Kp 5-1 derivative transformed with the non-self-transmissible pMMB207αß (an IncQ RSF1010 derivative) mobilized the vector into the parental strain with transfer frequencies of 10-3 transconjugants/donor. Identification of a TFSS in strain Kp 5-1 is significant since in other systems the mobilization capacity is involved in dissemination of plasmids that may confer antibiotic resistance and/or the delivery of virulence proteins into host cells, and thus may have an important role in the fitness of this strain as well. This is the first report that both compared and demonstrated functionality of a plasmid-harbored TFSS in a K. pneumoniae isolated from a N. viridula.

Potential for Nezara viridula (Hemiptera: Pentatomidae) to transmit bacterial and fungal pathogens into cotton bolls.

Recently, we showed that the southern green stink bug (SGSB), Nezara viridula (L.), can transmit Pantoea agglomerans (Ewing and Fife), an opportunistic bacterium, into green cotton bolls resulting in plant disease. Here, we hypothesized that our established model could be used to determine if the SGSB was a general, non-discriminate vector by using two other opportunistic bacterial pathogens of bolls (Pantoea ananatis [Serano] and Klebsiella pneumoniae [Schroeter]) and the known fungal pathogen Nematospora coryli (Peglion). Variants of P. ananatis (strain Pa-1R) and K. pneumoniae (strain Kp 5-1R) selected for rifampicin (Rif) resistance were used as bacterial opportunists. N. coryli was detected only from laboratory-reared SGSB directly exposed to the fungus. Both Pa-1R and Kp 5-1R were recovered from SGSB previously provided a contaminated food source (2 days), sterile food (5 days), and then harvested after being caged on bolls (2 days) at levels reaching 10(3) and 10(4) colony forming units (cfus) per insect, respectively. However, bolls caged with insects infected with Pa-1R or Kp 5-1R and with evidence of feeding did not become diseased nor were either opportunists detected from boll tissues. Insects infected with N. coryli transmitted the pathogen, which resulted in diseased bolls and fungi concentrations reached 10(6) cfus/g locule tissue at 2 weeks following the caging period. Notably, each of the three pathogens independently caused boll disease when mechanically inoculated using a needle puncture. Generally, these results suggest that cotton pathogen acquisition by the SGSB was not sufficient to determine whether the insects were disease vectors of the opportunists.

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

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 10(9), 10(9), and 10(3) 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 10(4) 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 10(8) 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.