A polymerase chain reaction assay for ascosporic inoculum of Sclerotinia species

Figures & data

Table 1 Fungal species and isolates used to test specificity of the SIG2598F/SIG2767R primers identified within the IGS region for Sclerotinia sclerotiorum detection.

Fungal species (isolate)	Host	Location	PCR detection
Sclerotinia sclerotiorum (DH1-07)	Tanacetum cinerariifolium	Sassafras, Tas, Australia	+
S. sclerotiorum (VPRI 22667)^1	Passiflora sp.	Victoria, Australia	+
S. sclerotiorum (64203)	T. cinerariifolium	East Devonport, Tas, Australia	+
S. minor (VPRI 21356)	Allium sp.	Victoria, Australia	+
S. trifoliorum (VPRI 22499)	Medicago sp.	Victoria, Australia	+
Alternaria alternata	T. cinerariifolium	Unknown	–
Botrytis cinerea (FG08)	T. cinerariifolium	Forth, Tas, Australia	–
B. cinerea (61705a)	Pyrethrum	Sassafras, Tas, Australia	–
B. cinerea (61705c)	Pyrethrum	Sassafras, Tas, Australia	–
Cladosporium cladosporioides	T. cinerariifolium	Unknown	–
Fusarium oxysporum	T. cinerariifolium	Unknown	–
Microsphaeropsis tanaceti (46009L-04)	T. cinerariifolium	Forth, Tas, Australia	–
Stagonosporopsis ligulicola var. inoxydabilis (58905)	T. cinerariifolium	Wesley Vale, Tas, Australia	–
Boeremia exigua var. exigua	T. cinerariifolium		–

Tas, Tasmania.

¹Victorian Plant Disease Herbarium, Melbourne, Australia.

Figure 1 PCR amplification using Sclerotinia sclerotiorum specific primers SIG2598F and SIG2767R to amplify 170-bp of DNA. M: 100-bp DNA ladder; Lane 1, Alternaria alternata; Lane 2, Cladosporium cladosporioides; Lane 3, Fusarium oxysporum; Lane 4, Microsphaeropsis tanaceti; Lane 5, Stagonosporopsis ligulicola var. inoxydabilis; Lane 6, Boeremia exigua var. exigua; Lane 7, Botrytis cinerea; Lane 8, Sclerotinia minor; Lane 9, Sclerotinia trifoliorum; Lane 10, Sclerotinia sclerotiorum; Lane 11, no DNA control.

Table 2 Number of positive PCR tests/total number of replicate samples for each Sclerotinia sclerotiorum ascospore concentration (with and without 10 mg soil).

Ascospores/DNA extraction	Ascospores/1 μLDNA (3 μL DNA)^1	Number of positive tests/total number of samples^2
		Ascospores	Ascospores + soil
0	0	0/10	0/10
10	0.2 (0.6)	10/10	7/10 (9/10)
25	0.5 (1.5)	10/10	9/10 (9/10)
50	1	10/10	10/10
100	2	10/10	10/10
500	10	10/10	10/10

¹Ascospores/PCR for 3 µL DNA presented parenthetically.

²Number of positive samples by PCR/total number of samples using 1 µL DNA. Numbers in parentheses are the total number of positive samples when samples that were negative with 1 µL DNA tested positive with 3 µL DNA added to the PCR reaction/total number of samples.

Table 3 Detection of ascosporic inoculum of Sclerotinia species on a rotating arm impaction spore trap by PCR amplification using primers SIG2598F and SIG2767R, and disease incidence of pods and plants in each of eight bean fields in Tasmania, Australia.

				Disease incidence (GS R4;%)^2
Field (Year)	Location	No. of positive air samples by PCR before first fungicide application/total samples^1	No. of positive air samples by PCR after first fungicide application/total samples^1	Pods	Plants
1 (2009)	Wesley Vale	1/5 (2/5)	6/6 (6/6)	1.4	2.5
2 (2009)	Mersey Lea	0/3 (0/3)	3/6 (5/6)	6.5	21.7
3 (2009)	Wesley Vale	2/2	5/8 (6/8)	0.0	0.3
4 (2009)	Forth	ND	1/7 (4/7)	0.1	0.3
5 (2010)	Wesley Vale	1/2 (1/2)	3/6 (3/6)	1.3	3.1
6 (2010)	Mersey Lea	2/6 (2/6)	1/6 (1/6)	5.9	30.8
7 (2010)	Mersey Lea	ND	1/7 (3/7)	0.2	0.3
8 (2010)	Wesley Vale	2/5 (2/5)	5/6 (6/6)	0.9	8.6

ND, not done.

¹Number of positive samples by PCR/total number of samples using 1 µL DNA. Numbers in parentheses are the total number of positive samples (by PCR)/total number of samples when those that tested negative with 1 µL DNA were tested again with 3 µL DNA.

²Growth stage R4: 50% of pods at maximum length (mid-pod set).