Initial stages of endophytic colonization by Metarhizium involves rhizoplane colonization

Here we assessed the time course of rhizoplane colonization by the endophytic insect pathogenic fungus Metarhizium robertsii. We describe a method of quantifying root colonization of bean plants by M. robertsii using quantitative polymerase chain reaction (qPCR). Results of this method were compared to the standard plate count method using colony-forming units (c.f.u.). Both the c.f.u. and qPCR methods were used to monitor the time-course of haricot bean (Phaseolus vulgaris) colonization by a strain of M. robertsii that expresses the green fluorescent protein (ARSEF 2575-GFP) for colony verification. There was a strong correlation between the results of the c.f.u. and qPCR methods, indicating that both methods are well suited for the determination of colonization of P. vulgaris roots by M. robertsii. Primers for a catalase gene (cat) amplified DNA from M. robertsii, M. brunneum and M. guizhouense. Primers for a nitrogen response-regulator (nrr) additionally detected M. acridum and M. flavoviride, whereas Metarhizium perilipin-like protein (mpl) primers were specific to M. robertsii alone. However, cat was the only target that specifically amplified Metarhizium in experiments utilizing non-sterile soil. Endophytic colonization of P. vulgaris at 60 days post-inoculation with M. robertsii was detected from surface-sterilized roots with more sensitivity using our qPCR technique over the c.f.u. method. Our results suggest that there is a prolonged period of rhizoplane colonization by Metarhizium with transient, low-level endophytic colonization of the root system of P. vulgaris that persists for the entirety of the plant life cycle.