Identification and comparison of biological characteristics and pathogenicity of different mating types of V. dahliae isolated from potato and sunflower.

Potato is one of the most important staple crops in the world. China is one of the leading producers of potatoes, but the industry faces soilborne diseases such as Verticillium wilt. Most potato planting areas in China rotate the crop with sunflower which is also highly susceptible to Verticillium wilt. The comparison of the biological characteristics and pathogenicity of different mating types of Verticillium dahliae isolated from potato and sunflower in the major planting regions in China is of great importance. This is to help unravel the diversity in V. dahliae population and the sudden increase in infected fields. The diseased samples collected were cultured on PDA and the growing colony of pathogen isolated. Molecular techniques using specific primers were used to identify the V. dahliae pathogens and their mating type of the isolates obtained from the diseased sunflower and potato plants as well as their planting materials. The data obtained revealed that the dominant mating type population in sunflower was MAT1-1, whiles that of potato was MAT1-2, but Race 2 was the only race type identified for all the samples. There was a significant presence of MAT1-1 isolates present in potatoes, which is a new trend. Conventional crop rotation farming using sunflower is causing an increasing prevalence of MAT1-1 and mating type shift of isolates in potato in these regions.

Fusarium incarnatum-equiseti Species Complex Causing Root Rot Disease on Leymus chinensis in China.

Leymus chinensis (Trin.) Tzvel. is a rhizomatous grass widely grown in the grasslands of Eurasia. With strong fertility and stress resistance, L. chinensis makes an excellent pasture and mowing grass, contributing to animal husbandry and thus playing an important role in the local economy of the northern grassland area in China (Baoyin et al. 2014). During August to September 2019, diseased roots of L. chinensis were collected from an artificially planted grassland (40°47'44" N, 111°43'58″ E, alt. 1049 m) in Shaerqin County, Hohhot, China. Infected plants were scattered across the field with disease incidence up to 2%. Symptoms observed were wilted plants and rotten roots. In order to identify the causal pathogen of root rot on L. chinensis, symptomatic pieces (5 × 5 mm) of grass roots were excised and surface sterilized with 75% ethanol for 3-5 s followed by 1% NaClO for 2-3 min, rinsed three times with sterile distilled water, and placed on water agar and incubated at 25°C for 3 days. The mycelia were cut and transferred onto potato dextrose agar (PDA) for subculture. A fungus was consistently isolated, and a strain, named LCH054, was obtained by hyphal tip culture. Culture developed as white and fluffy aerial mycelia, with diffused pink pigment on the reverse side of PDA after culturing at 25℃ for 7 days. A culture of LCH054 was transferred to carnation leaf agar (CLA) (Li et al. 2014) and incubated at 25°C for 10 days. Microconidia were absent but macroconidia were produced. Macroconidia were hyaline, sickle-shaped, and had 4 to 7 septa, 19.8 to 63.6 (mean 43.8) × 1.8 to 5.7 (mean 3.2) µm (n = 100). Chlamydospores were ellipsoidal or subglobose, with thick walls in clumps or chains. All morphological characteristics of LCH054 resembled Fusarium equiseti (Leslie and Summerell 2006). The primers of the internal transcribed spacer (ITS) region (White et al. 1990) and translation elongation factor 1α gene (TEF-1α) (O'Donnell et al. 1998) were used to amplify the isolate, and the fragments were sequenced. BLASTn search in the NCBI database using the ITS and TEF-1α sequences revealed 99 to 100% similarities with F. equiseti. BLAST analysis of the ITS and TEF-1α sequencies in the FUSARIUM-ID database showed them to have 99.21% (500 bp out of 504 bp) and 99.52% (622 bp out of 625 bp) similarities with the Fusarium incarnatum-equiseti species complex (FIESC) (strain NRRL 45997) (O'Donnell et al. 2009), respectively. The ITS and TEF1-α sequences were deposited in GenBank as accession numbers MT937067 and MT947530, respectively. The strain LCH054 was identified as a member of the FIESC based on morphological and molecular characteristics. For the pathogenicity test, one hundred of L. chinensis seeds were planted into five pots (12 cm [diameter]) × 15 cm [high]) and kept in a greenhouse under a 16-h photoperiod with temperatures of 20-25°C and 40% relative humidity. The conidial suspension of LCH054 was prepared by washing 7-day old fungal culture grown on CLA medium using sterile deionized water. Conidia were filtered through three layers of sterile cheese cloth, counted, and adjusted to 1 × 105 conidia/ml with a hemocytometer. Forty 1-month-old healthy plants (four pots) were inoculated with 400 ml of conidia suspension using the root drenching method, whereas the inoculum was replaced with 100 ml sterile water on control plants (one pot). Fourteen days after inoculation, all inoculated plants showed the typical symptoms of root rot identical to those observed in the field, whereas the control plants remained healthy. LCH054 was re-isolated from the inoculated plants and identified by the morphological and molecular approaches as described above. To the best of our knowledge, this is the first report of root rot caused by F. incarnatum-equiseti on L. chinensis in China as well as worldwide. The presence of the pathogen could cause significant economic losses in L. chinensis production. For this reason, strategies for the management and control of this disease should be developed and implemented.

Fungicide Treatments to Control Seed-borne Fungi of Sunflower Seeds.

: Seed-borne fungi in 69 sunflower cultivars were evaluated which comprised 52 confectionery and 17 oilseed types. Seed coats were placed on both NP-10 (Nonylphenol Ethoxylate based surfacant -10) and potato dextrose agar (PDA) media to culture fungi. The rate of contamination among the different varieties was calculated by counting seed coats with fungal colonies. The rate of contamination in the confectionary group (88%) was significantly (p ≤ 0.05) higher than in the oilseed group (71%). Of the 52 confectionery varieties, the dominant fungi recovered were Verticillium dahliae along with Alternaria spp., Fusarium spp., and Rhizopus spp., whereas the oilseed type varieties were contaminated with only V. dahliae. Molecular identification of fungal species via BLAST (Basic Alignment Search Tool) was performed on fungal sequences obtained from PCR (Polymerase Chain Reaction) analysis. The results included five Alternaria spp. that included Alternaria tenuissima, Alternaria alternata, Alternaria helianthiinficiens, Alternaria longipes, and Alternaria tamaricis, three Fusarium spp. such as Fusarium oxysporum, Fusarium incarnatum, and Fusarium proliferatum, and V. dahliae and Cladosporium cladosporioides. These were identified from pure fungal cultures recovered from seed coats. To efficiently control seed-borne fungi, four broad spectrum fungicides (carbendazim, triadimefon, caprio F-500, and flusilazole) were screened against V. dahliae isolate Gn3, which was isolated from a diseased LD 5009 sunflower plant. Flusilazole was selected based on its low half-maximal effective concentration value (EC50), 78.7 µg/mL. Seeds of diseased LD 5009 plants obtained from two different locations treated with formulated flusilazole fungicide at optimum parameters showed a significant (p ≤ 0.05) increase in seed germination and a decrease in contamination rate from 98% to less than 10%. The results affirmed that confectionery cultivars are much more susceptible to fungal contamination than oilseeds, and also that seed pretreatment is a suitable way to prevent the spread of soil- and seed-borne fungi in sunflower production.