A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

Summary

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

Abstract

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Introduction

Rhizoctonia represents a broad complex of fungi, of which Thanatephorus cucumeris (Frank) Donk (anamorph = Rhizoctonia solani Kühn) is the pathogen causing root rot and damping-off¹. Rhizoctonia solani is an aggressive pathogen and a saprophyte that can survive as sclerotia under adverse environmental conditions¹. As a result, it has a global distribution and can cause disease on wide range of plant hosts including Solanaceae, Fabaceae, Asteraceae, and Brassicaceae resulting in serious economic losses.

Compost has the capacity to harbor biocontrol agent....

Protocol

1. Prepare in Advance

1. Master fungal culture (test organism)
   1. Order from the American Type Culture Collection, Microbiology Collection⁹, by its teleomorph Thanatephorus cucumeris (Frank) (ATCC 10154) or MYA 4031.
   2. Alternatively: Collect local isolates as done by the authors. Seedlings of red radish (Raphanus sativus) work well as a bait plant; collect soil from a location known to have a history of damping-off or root rot caused by R. solani. Sow seeds in the soil and isolate from root lesions when seedlings are 3 to 4 weeks old.
      1. Remove seedlings from soil and rinse with t....

Results

Finished compost should be stable and mature, two terms that are often used interchangeably, so it can be safely packaged and transported, and not cause adverse effects during its end use⁴. Stability is a resistance to decomposition and is usually determined using indices of microbial activity. General measures of microbial respiration may measure compost stability but not necessarily disease suppression of an aggressive pathogen and saprophyte such as.......

Discussion

We know from previous research, that certain composts are effective at suppressing R. solani and that the suppressive effects are due to the microbes living in the compost, not the abiotic properties of compost^6,8. The use of autoclaving as a means to 'kill' microbiota has been criticized because it affects the carbon chemistry of the media¹⁰. We compared the use of autoclaving to vacuum filtration through Whatman No. 1 pa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
autoclavable narrow-neck glass conical flask	Fisher Scientific	10-040D	125-mL
autoclavable glass testtubes	Fisher Scientific	14-925J	25-mL
dehydrated granulated agar	Fisher Scientific	DF0145-17-0	500 g quantity
heat resistant gloves	Fisher Scientific	MEMGG1314WL	several brands available
Parafilm (strips of 2-3 cm wide)	Fisher Scientific	PM992 13-374-16	5 or 10 cm widths work
disposable polystyrene petri dishes	Fisher Scientific	R80116	comes in sleeves of 20/ea or cases of 500
dehydrated potato dextrose agar	Fisher Scientific	DF0013-15-8	comes in quantities of 100, 500 and 2000 grams
benchtop reciprocal shaker	Thomas Scientific	1227Y31	other models will work
water bath	ThermoScientific	S37363	5L general purpose
clear ruler, flat, at least 10 cm	Any		use metric rule
ATCC culture	American Type Culture Collection	ATCC 10154	teleomorph Thanatephorus cucumeris (Frank) (ATCC 10154) or MYA 4031;
lab tape	Fisher Scientific	15935	autoclavable and removable, 1" wide preferred
water resistant marker	office or scientific supply	Sharpie fine tip	write sample number on tape