Isolates of Cercospora kikuchii, a soybean (Glycine max) pathogen causing Cercospora leaf blight and purple seed stain, were tested to determine baseline sensitivities (n = 50) to selected quinone outside inhibitor (QoI) fungicides by conducting radial growth assays on fungicide-amended media. Baseline effective fungicide concentration to inhibit 50% of fungal radial growth (EC50) values were compared with EC50 values for isolates collected in 2011 (n = 50), 2012 (n = 50), and 2013 (n = 36) throughout soybean-producing areas in Louisiana. Median EC50 values for isolates subjected to QoI fungicides were significantly (P = 0.05) higher across all 3 years. Cross-resistance to QoI fungicides was observed in resistant isolates collected in 2011 to 2013. Discriminatory doses were developed for QoI fungicides to distinguish between sensitive and resistant isolates. On average, 89% of all isolates screened in 2011 to 2013 were resistant to QoI fungicides. At a discriminatory dose of thiophanate methyl (TM), a methyl benzimidazole carbamate (MBC) fungicide, at 5 μg/ml, resistance was detected in the 2000, 2011, 2012, and 2013 collections at 23, 38, 29, and 36%, respectively. Isolates exhibiting multiple resistance to QoI fungicides and TM also were detected in 2011, 2012, and 2013 at frequencies of 34, 26, and 31%, respectively. Based on these results, Cercospora leaf blight management strategies in Louisiana using solo applications of QoI or MBC fungicides in soybean should be reconsidered.
The fungus Simplicillium lanosoniveum was isolated from soybean leaves infected with Phakopsora pachyrhizi, the soybean rust pathogen, in Louisiana and Florida. The fungus did not grow or become established on leaf surfaces until uredinia erupted, but when soybean rust signs and symptoms were evident, S. lanosoniveum colonized leaves within 3 days and sporulated within 4 days. Development of new uredinia was suppressed by about fourfold when S. lanosoniveum colonized uredinia. In the presence of S. lanosoniveum, uredinia became increasingly red-brown, and urediniospores turned brown and germinated at very low rates. Assays using quantitative real time polymerase chain reaction revealed that the fungus colonized leaf surfaces when plants were infected with P. pachyrhizi, either in a latent stage of infection or when symptoms were present. However, when plants were inoculated before infection, there was no increase of DNA of S. lanosoniveum, suggesting that the pathogen must be present in order for the antagonist to become established on soybean leaf surfaces. We documented significantly lower amounts of DNA of P. pachyrhizi and lower disease severity when soybean leaves were colonized with S. lanosoniveum. These studies documented the mycophilic and disease-suppressive nature of S. lanosoniveum.
Cercospora leaf blight (CLB) of soybean, caused by Cercospora kikuchii, is a serious disease in the southern United States. A sensitive TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to specifically detect and quantify C. kikuchii in naturally infected soybean plants. The sensitivity was 1 pg of genomic DNA, which was equivalent to about 34 copies of genome of C. kikuchii. Using this qPCR assay, we documented a very long latent infection period for C. kikuchii in soybean leaves beginning at the V3 growth stage (as early as 22 days after planting). The levels of biomass of C. kikuchii remained low until R1, and a rapid increase was detected from the R2/R3 to R4/R5 growth stages shortly before the appearance of symptoms at R6. The efficacy of various fungicide regimens under field conditions also was evaluated over a 3-year period using this qPCR method. Our results showed that multiple fungicide applications beginning at R1 until late reproductive stages suppressed the development of C. kikuchii in leaves and delayed symptom expression. Different fungicide chemistries also had differential effects on the amount of latent infection and symptom expression during late reproductive growth stages.
Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits has been observed to survive for >34 years in stored melon and watermelon seeds. To better understand this remarkable longevity, we investigated the bacterium's tolerance to desiccation and the effect of bacterial localization in different watermelon seed tissues on its survival. We compared the ability of A. citrulli to tolerate desiccation on filter paper discs and on host (watermelon) and nonhost (cabbage, corn and tomato) seeds to two seedborne (Xanthomonas campestris pv. campestris and Pantoea stewartii subsp. stewartii) and one soilborne (Ralstonia solanacearum) plant-pathogenic bacteria. A. citrulli survival on dry filter paper (>12 weeks) was similar to that of X. campestris pv. campestris but longer than P. stewartii subsp. stewartii. Ralstonia solanacearum survived longer than all other bacteria tested. On all seeds tested, A. citrulli and X. campestris pv. campestris populations declined by 5 orders of magnitude after 12 weeks of incubation at 4°C and 50% relative humidity, while R. solanacearum populations declined by 3 orders. P. stewartii subsp. stewartii was not recovered after 12 weeks of incubation. To determine the effect of tissue localization on bacterial survival, watermelon seeds infested with A. citrulli by flower stigma inoculation (resulting in bacterial localization in the embryo/endosperm) or by ovary pericarp inoculations (resulting in bacterial localization under the testa) were treated with peroxyacetic acid or chlorine (Cl2) gas. Following these treatments, a significantly higher reduction in BFB seed-to-seedling transmission was observed for seeds generated by ovary pericarp inoculation (≥89.5%) than for those generated by stigma inoculation (≤76.5%) (P<0.05). Additionally, higher populations of A. citrulli survived when the bacteria were localized to the embryo/endosperm versus the seed coat, suggesting that tissue localization is important for bacterial survival in seed. This observation was confirmed when P. stewartii subsp. stewartii survived significantly longer in stigma-inoculated (embryo/endosperm-localized) watermelon seeds than in vacuum-infiltrated (testa-localized) seeds. Based on these results we conclude that A. citrulli cells are not intrinsically tolerant to desiccation and that localization of the bacterium to testa tissues does not enhance A. citrulli survival. In contrast, it is likely that embryo/endosperm localization enhances the survival of A. citrulli and other bacteria in seeds.
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