. 2021 Apr 25;9(5):920. doi: 10.3390/microorganisms9050920

Table 3.

Yakima soil analysis for the detection of fungicide resistant T. harzianum (ThTMS3a).

Sampling Time	Field Plot	Sample Analyzed	Non-Selection Media		Selection Media
			-Fungicides		+Fungicides
			Trich. spp.	Other Fungi	Trich. spp.	Other Fungi	T. harzianum
Pre-planting 1 month	Untreated	soil	+	++	-	-	-
Pre-planting 1 month	ThMS3a	soil	+	++	-	-	-
Post-planting 2 weeks	Untreated	soil at plant base	+	++	-	-	-
Post-planting 2 weeks	ThMS3a	soil at plant base	+	++	+	-	+
Post-planting 2 and 3 months	Untreated	soil at plant base	+	++	-	-	-
Post-planting 2 and 3 months	ThMS3a	soil at plant base	+	++	-	-	-
Post-harvest 4 months	Untreated	soil at plant base	+	++	-	-	-
Post-harvest 4 months	ThMS3a	soil at plant base	+	++	-	-	-
	Untreated	non-sterilized roots and crowns	-	+	-	-	-
	ThMS3a	non-sterilized roots and crowns	-	+	-	-	-
1 year post-harvest	Untreated	surface sterilized roots and crowns	-	-	-	-	-
1 year post-harvest	ThMS3a	surface sterilized roots and crowns	-	-	-	-	-

Two plots (6 ha) adjacent to each other were chosen for field studies to assess for the presence of ThTMS3a in soils and plant tissues throughout the year. One month prior to planting, soils were randomly assayed in 10 locations per plot. 20 cm soil core samples surrounding the plant root rhizosphere (N = 10) were collected at one-month pre-planting, 2 weeks, 2- and 3-months post-planting, and a final soil sampling 4 months post-harvest. Collectively, 10 non-selection (0.1 × PDA plate supplemented with 100 μg/mL of ampicillin and chloramphenicol) and selection media plates (media containing thiabendazole, hymexazol, and tebuconazole) representing <1000 fungal CFU were assayed for soil samples of each time point. Non-selection plates revealed >10% of the fungal CFU as Trichoderma spp. (Trich. spp.) indicating its presence in low numbers (+); compared to collectively the high presence (++; <90%) of other common soil fungi (Aspergillus, Fusarium, Alternaria, Mucor, Penicillium, Curvularia, and Colletotrichum spp.). A low level (+, >10%) of Trichoderma spp. isolates microscopically verified as T. harzianum were detected from rhizosphere soils from the base of plants collected 2 weeks post-plating. No other fungi were detected on selection media for any other soil samples. One-year post-harvest, plant crown and upper roots (N = 10) were collected from untreated and ThMS3a treated plots. Tissues were surface sterilized or not, and six 5 cm tissue samples representative of the roots and crowns for each sample, placed on non-selection and selection media plates. Growth of Trichoderma was not observed in any of the samples but other fungal species were present in soils and grew out of non-sterilized plant tissues plated on non-selection plates. The samples were scored for the presence (+) or absence (−) of fungi. CFU was not able to be determined as growth occurred out of plant tissues. Colors represent different treatments and organisms isolated from samples.