Michael B. Blackburn

Q-type C2H2 zinc finger proteins (ZFPs) play a role in plant stress response. Overexpression of individual ZFPs enhances tolerance to abiotic stresses such as drought or salt. Overexpression of ZFP, Zat18 enhances tolerance to drought, whereas plants with lower amounts are less resilient. The effect of Zat18 loss on gene expression has not been characterized. Overexpression of an ortholog of Zat18 in potato, StZFP2 enhances tolerance to infestation by Manduca sexta. This work focuses on two Zat18 mutant lines, a knockout and knockdown. The two lines differ in response to infestation. Typically, a chewing insect induces the jasmonic acid (JA) pathway. While infestation induces JA pathway genes, Zat18 mutant lines downregulate abscisic acid signaling and induce ethylene signaling genes ERF1 and EBF2 as well as salicylic acid response gene NPR3. Knockdown Zat18, cause far more changes in the infestation response compared with Col-0 or the Zat18 knockout line.

In recent years, several studies have examined the gut microbiome of lepidopteran larvae and how factors such as host plant affect it, and in turn, how gut bacteria affect host plant responses to herbivory. In addition, other studies have detailed how secretions of the labial (salivary) glands can alter host plant defense responses. We examined the gut microbiome of the cabbage looper (Trichoplusia ni) feeding on collards (Brassica oleracea) and separately analyzed the microbiomes of various organs that open directly into the alimentary canal, including the labial glands, mandibular glands, and the Malpighian tubules. In this study, the gut microbiome of T. ni was found to be generally consistent with those of other lepidopteran larvae in prior studies. The greatest diversity of bacteria appeared in the Firmicutes, Actinobacteria, Proteobacteria, and Bacteriodetes. Well‐represented genera included Staphylococcus, Streptococcus, Corynebacterium, Pseudomonas, Diaphorobacter, Methylobacterium, Flavobacterium, and Cloacibacterium. Across all organs, two amplicon sequence variants (ASVs) associated with the genera Diaphorobacter and Cloacibacterium appeared to be most abundant. In terms of the most prevalent ASVs, the alimentary canal, Malpighian tubules, and mandibular glands appeared to have similar complements of bacteria, with relatively few significant differences evident. However, aside from the Diaphorobacter and Cloacibacterium ASVs common to all the organs, the labial glands appeared to possess a distinctive complement of bacteria which was absent or poorly represented in the other organs. Among these were representatives of the Pseudomonas, Flavobacterium, Caulobacterium, Anaerococcus, and Methylobacterium. These results suggest that the labial glands present bacteria with different selective pressures than those occurring in the mandibular gland, Malpighian tubules and the alimentary canal. Given the documented effects that labial gland secretions and the gut microbiome can exert on host plant defenses, the effects exerted by the bacteria inhabiting the labial glands themselves deserve further study.

Two isolates of Gram-reaction-negative, motile, violet-pigmented bacteria were isolated from a small pool in marshland near the mouth of the Nanticoke River in Maryland, USA. The isolates IIBBL 257-1(T) and IIBBL 257-2 had identical 16S rRNA gene sequences as determined by PCR, and highly similar fatty acid and biochemical profiles. The 16S rRNA gene sequences indicated the isolates belonged to the genus Chromobacterium. Genomic sequencing of IIBBL 257-1(T) revealed a genome of 4.27 Mb, with a G+C content of 63.6%. Whole genome comparisons with other members of the Chromobacterium using JSpecies and the genome blast distance phylogeny approach indicated that among described species, IIBBL 257-1(T) was most closely related to C. amazonense and C. phragmitis. Comparison of the IIBBL 257-1(T) genome with those of type strains of these species resulted in ANIb and dDDH values of ca. 85 and 30%, respectively, for both. These results demonstrate that IIBBL 257-1(T) and IIBBL 257-2 represent a new taxon within the genus Chromobacterium. We propose the name Chromobacterium paludis sp. nov. for this taxon; the type strain is IIBBL 257-1(T) (=NRRL B-65555(T)=JCM 33770(T)).

Several isolates of the recently described bacterial species Chromobacterium phragmitis Blackburn et al. were obtained from water collected from low-salinity tidal marshes in Maryland and Virginia. Bacteria were cultured in a liquid medium and applied to artificial diets in the laboratory. One of two Maryland isolates, IIBBL 113-1, was highly toxic to larvae of the cabbage looper Trichoplusia ni (Hübner) and the diamondback moth, Plutella xylostella (L.). The other Maryland isolate, IIBBL 112-1T, and the Virginia isolates were less toxic to these species. Maryland isolates were toxic to larvae of the seedcorn maggot Delia platura (Meigen), while a Virginia isolate, IIBBL 274-1, was of intermediate toxicity against D. platura. The C. phragmitis isolate IIBBL 113-1 was toxic to adults of the red flour beetle Tribolium castaneum (Herbst), while other C. phragmitis isolates had no activity against this species.

Thirteen isolates of Gram-stain-negative, motile, violet-pigmented bacteria were isolated from marshes along tidal portions of the Potomac and James rivers in Maryland and Virginia, USA, respectively. 16S rRNA gene sequences and fatty acid analysis revealed a high degree of relatedness among the isolates, and genomic sequencing of two isolates, IIBBL 112-1T and IIBBL 274-1 (from the Potomac and James rivers, respectively), revealed highly similar genomic sequences, with a blast-based average nucleotide identity (ANIb) of ca. 98.7 %. Phylogenetic analysis of 16S rRNA gene sequences suggested that the species most highly related to IIBBL 112-1T were Chromobacterium amazonense , Chromobacterium subtsugae and Chromobacterium sphagni . However, deletion of a 25-nucleotide sequence that may have been horizontally acquired by both IIBBL 112-1T and C. amazonense resulted in a substantially different analysis; in the latter case, the species nearest IIBBL 112-1T were Chromobacterium violaceum , Chromobacterium vaccinii and Chromobacterium piscinae . Whole-genome alignments between either IIBBL 112-1T or IIBBL 274-1 and the type strains of C. vaccinii or C. violaceum resulted in ANIb values in the range of ca. 87 %, while alignment with C. amazonense CBMAI 310T resulted in an ANIb of ca. 83 %. Collectively, these data demonstrate that IIBBL 112-1T and IIBBL 274-1 represent a new taxon within the genus Chromobacterium . We propose the name Chromobacterium phragmitis sp. nov. for this taxon; the type strain is IIBBL 112-1T (=NRRL B-67132T=JCM 31884T).

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera), is an important pest of citriculture. The ACP vectors a bacterium that causes huanglongbing (HLB), a devastating and incurable disease of citrus. The bacterium Bacillus thuringiensis (Bt) produces multiple toxins with activity against a diverse range of insects. In efforts to provide additional control methods for the ACP vector of HLB, we identified pesticidal proteins derived from Bt for toxicity against ACP. The trypsin proteolytic profiles of strain-derived toxins were characterized. Strain IBL-00200, one of six strains with toxins shown to have basal activity against ACP was selected for liquid chromatography-mass spectrometry (LC-MS/MS) identification of the individual Cry toxins expressed. Toxicity assays with individual toxins derived from IBL-00200 were then performed. The activated form of the Cry toxins Cry1Ab and Cry1Ba were toxic to ACP with LC₅₀ values of approximately 120 µg/mL. Disruption of the midgut epithelium was associated with the toxicity of both the IBL-00200-derived toxin mixture, and with Cry1Ba. With further optimization of the efficacy of Cry1Ab and Cry1Ba, these toxins may have practical utility against ACP. Bt toxins with activity against ACP may provide an additional tool for management of ACP and the associated HLB disease, thereby providing a more sustainable and environmentally benign approach than repeated application of broad-spectrum insecticides.

Several isolates of the recently described bacterial species Chromobacterium sphagni Blackburn et al. were obtained from water collected from Sphagnum bogs in West Virginia and Maine. Bacterial isolates were cultured in a liquid medium and applied to artificial insect diets in the laboratory. The new isolates were toxic to larvae of the gypsy moth, Lymantria dispar (L.), and the diamondback moth, Plutella xylostella (L.), but were not toxic to larvae of the seedcorn maggot, Delia platura (Meigen), or adults of the red flour beetle, Tribolium castaneum (Herbst).

New Chromobacterium sp. strains were isolated from water collected from several localities in the eastern United States and were subsequently identified as the species Chromobacterium vaccinii Soby et al. Bacteria were cultured in a liquid medium and applied to artificial insect diets in the laboratory. All of the recently discovered C. vaccinii isolates tested were toxic to larvae of the seedcorn maggot, Delia platura (Meigen), but had little activity against larvae of the diamondback moth, Plutella xylostella (L.). None of the new isolates of C. vaccinii were toxic to adults of the red flour beetle, Tribolium castaneum (Herbst).

Sixteen isolates of Gram-reaction-negative, motile, violet-pigmented bacteria were isolated from Sphagnum bogs in West Virginia and Maine, USA. 16S rRNA gene sequences and fatty acid analysis revealed a high degree of relatedness among the isolates, and genome sequencing of two isolates, IIBBL 14B-1T and IIBBL 37-2 (from West Virginia and Maine, respectively), revealed highly similar genomic sequences. The average nucleotide identity (gANI) calculated for these two isolates was found to be in excess of 99 %, but did not exceed 88 % when comparing either isolate with genomic sequences of Chromobacterium violaceum ATCC 12472T, C. haemolyticum DSM 19808T, C. piscinae ND17, C. subtsugae PRAA4-1T, C. vaccinii MWU205T or C. amazonense CBMAI 310T. Collectively, gANI and 16S rRNA gene sequence comparisons suggested that isolates IIBBL 14B-1T and IIBBL 37-2 were most closely related to C. subtsugae , but represented a distinct species. We propose the name Chromobacterium sphagni sp. nov. for this taxon; the type strain is IIBBL 14B-1T (=NRRL B-67130T=JCM 31882T).