Item

Brevibacillus laterosporus as a potential bio-control agent of the diamondback moth and other insects

Ormskirk, Mildred Marsha
Date
2017
Type
Thesis
Fields of Research
ANZSRC::060599 Microbiology not elsewhere classified
Abstract
Two New Zealand strains of Brevibacillus laterosporus (Bl), Bl 1821 and 1951, originally isolated from brassica seeds, displayed high activity toward the larvae of the diamondback moth (DBM), Plutella xylostella, and also toward mosquito larvae in bioassays. Research was commenced to develop Bl 1821 and 1951 as biocontrol agents (BCAs) of the DBM and other insects, and this study contributed to this larger aim. Novel Crystal toxin-like (Cry-like) encoding genes were discovered in the strains of Bl and were hypothesised to be responsible for the DBM and mosquitocidal activity of the strains. Bl 1821 produced crystalline parasporal bodies in the sporangia, and these were hypothesised to contain the DBM toxins. The main objectives of this study were to: 1) Localise the main DBM toxicity in the native cultures of the strains by the separation of spores and crystals by gradient centrifugation and, in the case of Bl 1951, by a separation of spores and culture supernatant by conventional centrifugation, and to identify any plasmids of the strains; 2) Confirm DBM toxicity of the putative toxin Cry-like proteins of the Bl 1821 and 1951 by heterologous expression in Escherichia coli, purification of the recombinant proteins and subsequent larval DBM bioassays; 3) Identify the DBM protein toxins in native cultures of Bl 1951 by ESI-mass spectrometry; 4) Study the endophytic potential of Bl 1821 and 1951 in cabbage plants and, in the case of Bl 1951, in a brassica endophyte field trial with cabbage plants; 5) Assess the field performance of unformulated Bl 1821 and 1951 sporulated cultures in brassica field trials. A loss of virulence affected the strains during the project in both the laboratory and field applications. The cultures showed common signs of a bacteriophage infection. Therefore, two additional objectives were included: 1) To identify the cause of the loss of virulence toward the DBM of Bl 1821 and 1951; and 2) To restore the virulence of the strains. No DBM activity was displayed by the vegetative cells and spores of Bl 1821 and 1951, or in the case of Bl 1821, in the parasporal crystalline bodies. A few plasmids were identified in Bl 1821, 1951 and an additional strain, Bl RSP, isolated from potatoes. No DBM activity was displayed by the Cry27-like and Cry35-like recombinant proteins as inclusion bodies (IBs), or by the Cry35-like refolded proteins. The main DBM activity of Bl 1951 was located in the culture supernatant (CSN) and a surface layer (S-layer) protein, derived from the CSN, was identified as a putative DBM toxin. Bio-informatic analysis also revealed the presence of two putative accessory virulent genes, collocated to the S-layer protein-encoding gene. Both Bl 1821 and 1951 were identified as putative endophytes of cabbage in a pot trial, and Bl 1951 was identified as a putative endophyte of cabbage after field application. Both strains significantly reduced insect leaf damage in cabbage field trials compared to untreated plants and in two trials the performance of Bl 1821 was comparable to DipelDF. A Bl 1951 field trial treatment showed high activity toward larvae of the white butterfly (Pieris rapae) comparable to DipelDF in a laboratory bioassay. The loss of virulence displayed by the strains in laboratory and field conditions over the course of the study was explained by the discovery of a Tectivirus-like phage, which was identified in a native culture of Bl 1821 by Transmission Electron Microscopy, and may exist as a plasmodial prophage in Bl. The activity of the strains was partially recovered after a change of culturing conditions and was related to the addition of glucose to the growth medium as an extra carbon source for cellular respiration. Bl RSP showed high larval DBM mortality, up to 88%, by day 5. This is the first report of Bl RSP DBM toxicity. The strains shared the same ~7.5 kb plasmid and Bl 1821 and RSP shared a >15 kb plasmid, which may be involved in the virulence of the strain or may represent the plasmid DNA of the Tectivirus-like phage. The putative DBM toxicity of the S-layer protein of Bl 1951 needs to be confirmed by heterologous expression and gene knockout studies. The same gene was also identified in Bl 1821 and RSP and may be responsible for the DBM toxicity displayed by these strains as well. The S-layer protein may represent a novel class of toxins that can be exploited for DBM control. In conclusion, Bl 1821 and 1951 show potential as biocontrol agents of the DBM, and in the case of Bl 1951, also of the white butterfly.
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