Field studies using a recombinant mycoinsecticide (Metarhizium anisopliae) reveal that it is rhizosphere competent

Abstract

In the summer of 2000, we released genetically altered insect-pathogenic fungi onto a plot of cabbages at a field site on the Upper Marlboro Research Station, Md. The transformed derivatives of Metarhizium anisopliae ARSEF 1080, designated GPMa and GMa, carried the Aequorea victoria green fluorescent protein (gfp) gene alone (GMa) or with additional protease genes (Pr1) (GPMa). The study (i) confirmed the utility of gfp for monitoring pathogen strains in field populations over time, (ii) demonstrated little dissemination of transgenic strains and produced no evidence of transmission by nontarget insects, (iii) found that recombinant fungi were genetically stable over 1 year under field conditions, and (iv) determined that deployment of the transgenic strains did not depress the culturable indigenous fungal microflora. The major point of the study was to monitor the fate (survivorship) of transformants under field conditions. In nonrhizosphere soil, the amount of GMa decreased from 10(5) propagules/g at depths of 0 to 2 cm to 10(3) propagules/g after several months. However, the densities of GMa remained at 10(5) propagules/g in the inner rhizosphere, demonstrating that rhizospheric soils are a potential reservoir for M. anisopliae. These results place a sharp focus on the biology of the soil/root interphase as a site where plants, insects, and pathogens interact to determine fungal biocontrol efficacy, cycling, and survival. However, the rhizospheric effect was less marked for GPMa, and overall it showed reduced persistence in soils than did GMa.

FIG. 1.

Mean soil titers of propagules of Paecilomyces farinosus, (▴) Penicillium spp. (•), and Aureobasidium pullulans (▪) in the application area treated with M. anisopliae GMa. Soil samples were taken within 1 cm from cabbage plant roots at depths of 0 to 2 cm, and fungal propagules were quantified on Veens medium minus cycloheximide. Error bars indicate SD.

FIG. 2.

Changes in the soil titer of GMa (▾) and GPMa (•), over 300 days, within 1 cm of cabbage plant roots at depths of 0 to 2 cm. Spore count data were transformed to the log scale. The lines represent the model outcome of the population decline of GMa (dashed line) and GPMA (solid line). The analysis was conducted using Proc MIXED, SAS. Error bars indicate SD.

FIG. 3.

Effect of proximity to cabbage roots on the persistence of M. anisopliae under field conditions. Mean soil titers of propagules of M. anisopliae GMa and GPMa at depths of 0 to 2 cm are shown. Error bars indicate SD.

FIG. 4.

Mean number of propagules of M. anisopliae GMa at different depths in the outer (▪) and inner (□) rhizosphere of cabbage roots, 4 months after application. Error bars indicate SD.

FIG. 5.

UV micrographs of 2- to 5-cm-deep cabbage roots from application area 2 (4 months after spraying with GMa) placed on Veens medium for 48 h and showing growth by fluorescent M. anisopliae. The roots were shaken free of rhizosphere soil (A) or, in addition, ultrasonicated and subjected to a series of 10 water washes (B), which removed the inner rhizosphere and most but not all fungal propagules (indicated by arrows).