Streptomyces and Bacillus species utilize volatile organic compounds to impact Fusarium oxysporum f.sp. vasinfectum race 4 (Fov4) virulence and suppress Fusarium wilt in Pima cotton

Emergence of a highly virulent Fusarium oxysporum f.sp. vasinfectum race 4 (Fov4) with aggressiveness towards Pima cotton (Gossypium barbadense) has raised significant concern for cotton producers while revealing challenges in soil-borne cotton disease management strategies which rely heavily on crop resistance and chemical controls. An alternative management approach uses antagonistic bacteria as biocontrol agents against Fov4. Initial studies showed a unique combination of bacteria Bacillus Rz141 and Streptomyces HC658 isolates displayed a mutualistic relationship capable of altering Fov4 growth. Notably, experimental design placed Fov4 between each isolate preventing direct physical contact of bacterial colonies. These observations led us to hypothesize that bacterial volatile organic compounds (VOCs) impact the growth and virulence of Fov4. Ensuring physical separation, I-plate cultures showed Rz141 had a VOC inhibition of 24%. Similarly, physically separated cultures of Rz141 and HC658 showed slight increase in VOC inhibition, 26% with some loss of Fov4 pigmentation. Pathogenicity assays where Fov4-infected Pima cotton was exposed to VOCs from physically separated Rz141 and HC658 showed VOCs can suppress Fov4 infection and reduce tissue darkening. Our results provide evidence that rhizosphere bacteria can use VOCs as a communication tool impacting fungal physiology and virulence, and ultimately Fov4-cotton interactions without direct physical contact.

An in vitro co-culture system for rapid differential response to Fusarium oxysporum f. sp. vasinfectum race 4 in three cotton cultivars

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a devastating fungus pathogen that causes Fusarium wilt in both domesticated cotton species, Gossypium hirsutum (Upland) and G. barbadense (Pima). Greenhouse and field-based pathogenicity assays can be a challenge due to non-uniform inoculum levels, the presence of endophytes, and varying environmental factors. Therefore, an in vitro co-culture system was designed to support the growth of both domesticated cotton species and FOV4 using an inert polyphenolic foam substrate with a liquid medium. A Fusarium wilt-susceptible Pima cotton cultivar, G. barbadense ‘GB1031’, a highly resistant Pima cotton cultivar, G. barbadense ‘DP348RF’, and a susceptible Upland cotton cultivar, G. hirsutum ‘TM-1’, were evaluated for 30 days during co-culture with FOV4 in this foam-based system. Thirty days after inoculation, disease symptoms were more severe in both the susceptible cultivars, which displayed higher percentages of foliar damage, and greater plant mortality, than observed in ‘DP348RF’, the resistant Pima cotton cultivar. This foam-based in vitro system may be useful for screening cotton germplasm for resistance to a variety of fungus pathogens and to facilitate the study of biotic interactions in domesticated cotton species under controlled environmental conditions.

Tolerance of Pima and Upland cotton to trifloxysulfuron (Envoke) herbicide under field conditions

Trifloxysulfuron (Envoke) is an acetolactate synthase-inhibitor herbicide and can be used to control many broadleaf weeds and nutsedges in cotton production. However, there is a lack of information on genotypic variation in response to the herbicide. In this field study, 60 Pima (Gossypium barbadense L.) lines, 122 Upland (G. hirsutum L.) lines, and 9 Upland × Pima segregating populations were divided into five tests (18A, 18B, 18G, 18RB, and 18HQ) to evaluate trifloxysulfuron tolerance at the 7-true leaf stage (42 days after planting) under the same field conditions in 2018. Across the five tests, Pima cotton genotypes tested in this study did not show any visual crop injury based on percentage of plants with chlorosis at 6 days after treatment (DAT), indicating consistent and high levels of trifloxysulfuron tolerance. However, the response to trifloxysulfuron within Upland cotton is highly variable. While Upland cotton is overall more sensitive to trifloxysulfuron with crop injury up to 80% than Pima cotton, 19 lines had injury below 5% including one line with no visual injury, and 19 lines had injury between 5% and 10%. In test 18HQ with 15 transgenic Upland cultivars and 17 non-transgenic Upland lines, the analysis of variance detected a significant genotypic difference. The broad-sense heritability estimates for trifloxysulfuron tolerance based on crop injury at 6 DAT was 0.555, suggesting that trifloxysulfuron tolerance in Upland cotton is moderately heritable. This study represents the first report that Pima cotton and many Upland cotton lines are highly tolerant to trifloxysulfuron with no or little crop injury.

Soil-free pathogenicity assay of Fusarium oxysporum f.sp. vasinfectum race 4 on Pima cotton (Gossypium barbadense) seedlings

One of the key early season diseases of cotton is Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum (Fov). Recent emergence of highly virulent Fov race 4 (Fov4) and its aggressiveness toward Gossypium barbadense (pima) cultivars are raising significant concerns for the US cotton industry. One of the key challenges in studying Fov4 virulence and cotton Fusarium wilt pathogenesis is establishing a disease assay strategy that can help researcher overcome several technical challenges, including efficient infection and highly reproducible and consistent symptom development. Here, we have developed a small-scale, soil-free Fusarium wilt disease assay that can complement conventional assays with faster symptom development and high reproducibility in infected pima cotton seedlings. Our data showed statistically significant differences (p<0.0001) between Fov4-infected and non-infected pima cotton at 4 and 6 days post inoculation (dpi) when compared to control experiments. At 6 dpi, longitudinal observations under magnification showed Fov4 colonization in primary xylem of infected plants, which is a common symptom observed in Fov4 triggered Fusarium wilt in pima cotton. While this is an artificial assay system, this soil-free disease testing strategy can offer another strategy to supplement current assays when studying pathogen-host interaction in soil-borne diseases.