A new method for identification and enumeration of microorganisms with potential for suppressing fungal plant pathogens

Microorganisms with ability to suppress disease causing fungi and insect-pests are potentially important alternatives to chemical pesticides and have been reported by many researchers. Farmers producing organic food reported reduced incidence of diseases and insect pests. The organic farmers in Karnataka, India, when visited in 1998, also stated that their crops generally had less disease incidence than those of their neighbors following mainstream agriculture. It was guessed that soil of organic farms and some of the alternatives to chemicals used by them had high population of microorganisms that suppressed the growth of disease causing fungi. An urge to verify this led us to devise a laboratory method of rapidly identifying microorganisms with potential to suppress plant pathogenic fungi (antagonistic microorganisms) in the presence of other microorganisms and is described here.

The usual method of identification of such microorganisms involves isolation of a large number of microorganisms from a target source followed by screening against a target pathogen using Dual Culture method (Figure 1). This method does not allow a direct enumeration of antagonists present in the source material.

Figure 1. Four different antagonists under evaluation, using Dual Culture method.

A Two-Layer method was developed that allowed counting of the total culturable and antagonistic microorganisms. Fusarium solani (causes black root rot of chickpea) was used as the test fungus (pathogen). A suspension of this fungus was spread plated on a quarter-strength (¼) potato dextrose agar (PDA). After about 4-hour drying in a laminar-flow, each plate was further layered with (2-3 mm thick) ¼ PDA on which 0.1 mL of appropriate dilution of a soil sample (or any other source material) was spread plated. Bacteria appeared within 24 h and were counted. In about four days F. solani grew through the upper layer and in another four days grew on all the microbial colonies except few per plate. These colonies had a halo (apparently devoid of fungal growth) around them and were counted as antagonistic microorganisms. Microorganisms from the colonies with widest band (>2 mm) of halo around them were isolated and purified for further studies. (Figure2) has a series of dilution plates from such a study.

Figure 2. Petri-plates of the Two-Layer method at day nine.

Population of total bacteria in six specific composts (called Biodynamic or BD preparations by organic farmers using these composts) ranged from 3.45 log10 (BD 502) to 8.59 log10 (BD 504) per g of dry materials. Population of antagonistic bacteria was counted in three of the six composts and ranged from 3.24 log10 (BD 502) to 6.90 log10 (BD 500). Thirteen to 36% of the colonies on the plates used for enumeration of the total population were antagonistic to the test pathogenic fungus. Even termitaria soil had large population of total bacteria (6.54 log10 per g) and total fungi (4.72 log10 per g). Of these, 5.01 log10 per g of bacteria and 2.74 log10 per g of fungi were antagonistic to F solani.

A collection of 67 bacterial isolates was assembled from the 6 BD preparations and three other sources. All these isolates were screened for their ability to suppress four fungi causing different diseases (dry root rot of chickpea, collar rot of chickpea, fusarium wilt of chickpea and aflatoxin of groundnut) using Dual Culture method. Seventeen of the 67 isolates (25%) suppressed at least one of the four fungi, in addition to the F. solani (as measured by the Two-Layer method).

The Two-Layer method allowed counting the population of antagonists in compost samples that was not feasible with the Dual Culture method. Also, we could count the total microbial population (culturable) in compost samples. As was confirmed in unpublished subsequent studies, total count of microorganisms by the Two-Layer method matched well with the counts on PDA plates. High population of antagonists was indeed present (Table 1) in the six different composts used by the organic farmers in Karnataka and Tamil Nadu, India and may be the basis of their crops having low incidence of diseases and insect pests. This will need confirmatory studies.

Table 1. Microbial population in different composts.

	Total microbial Population (log10 g-1)		Population of antagonists (log10 g-1)		Total no. of colonies on 2 replicate plates used for counting (% colonies with hollow)
Source	Bacteria	Fungi	Bacteria	Fungi
BD 500	7.35	*	6.90¶	*	45 (36)
BD 502	3.45	5.30	3.24¶	3.98¶	97 (26)
BD 503	7.10	*	NC	NC	152 (NC)
BD 504	8.59	*	NC	NC	203 (NC)
BD 505	6.63	*	NC	NC	137 (NC)
BD 506	4.91	4.26	4.28	4.22	527 (13)
Backyard compost	6.65	*	5.69¶	*	90 (11)
Billbergia spp.	6.52	5.60¶	6.08¶	5.60¶	111 (12)
Termite soil	6.54	4.72¶	5.01¶	2.74¶	81 (32)
SE	0.080	0.059	0.409	1.328	NR
NC = Not counted, NR = Not relevant, *= the dilutions used for spread plated did not have any fungal colony, BD = Biodynamic preparation, SE = effective standard error. ¶ = Plates with <30 colonies were used for counting the population and therefore these data should be taken with caution. The plates of dilutions lower than the ones used in collecting the data, were not used because of poor expression of hollow (or of fungal colonies) due to very high population of other microorganisms.

The method allows use of two different media in the upper and lower layers. In unpublished studies, we used Glucose-Cassamino-acids Yeast Extract (GCY) medium in the lower layer for growing Aspergillus flavus and ¼ PDA as upper layer. This combination of media was used for identification and enumeration of antagonists in groundnut rhizosphere soil samples.

For a dependable count of microorganisms in a sample one should use plates having 30 to 300 colonies (a norm). It sometimes required a repeat to fulfill this condition because the population of antagonists will only be a fraction of the total count on a given plate. And in some other cases it was still not feasible to fulfill this condition because the high population of other microorganisms (not having halo) per plate did not allow expression of antagonists. This seems to have resulted in high coefficient of variation (CV%) in the relevant data. The variation in the case of the counts of antagonistic fungi and antagonistic actinomycetes in all the samples was generally higher than in case of bacteria (data not presented).

Most of the 67 isolates that were picked up due to having a halo with the Two Layer method did not show a clear zone with F. solani when evaluated using a Dual Culture method. However, the fungus F. solani did not grow over the colonies of these strains. It seems that the mechanism of action of the isolates (that showed halo in the Two-Layer method but no zone formation in the Dual Culture method) is different than secretion of some toxin and needs further studies.

Acknowledgements:

We thank Drs S D Singh and R P Thakur for providing the fungal cultures used in the study and for comments on the previous version of the paper.


Legends to figures:

Figure 1. Four different antagonists under evaluation, using Dual Culture method. The target plant pathogenic fungus is grown on¼ PDA, in the middle of the plate, and the four potential antagonists are spotted at equal distance from the center of the fungus.

Figure 2. Petri-plates of the Two-Layer method at day nine. The upper three plates are of a series of dilutions (from left, 10-4to 10-6) of BD 500 and the lower three plates are of the same dilutions of Backyard compost. Note the fungus F. solani (plated on the lower layer) has grown through the upper layer of ¼ PDA and has covered most of the organisms except few (visibly having a halo).

For more information please contact:

Dr Rupela O P
Scientist (Soil Biology),
ICRISAT-Patancheru 502 324.