Interaction of Fusarium Wilt Race 4 with Root-Knot Nematode Increases Disease Severity in Cotton.

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum, is a severe disease of cotton (Gossypium spp.). Strains of the wilt pathogen in the United States, such as race 1, require the presence of nematodes such as southern root-knot nematode (Meloidogyne incognita) to cause appreciable disease. The exception is the race 4 strain of the wilt pathogen, which can attack cotton without concomitant infection by plant-parasitic nematodes and was first identified in California in 2001 and in Texas and New Mexico since 2017. The effects of the interaction between M. incognita and race 1 or race 4 on wilt severity and nematode reproduction on two Gossypium hirsutum cultivars, Acala 44 and FM 966, and a G. barbadense cultivar, Pima S-4, were directly compared in growth chamber assays. All three cultivars were susceptible to M. incognita. Suppression of nematode reproduction by the wilt pathogen was detected only for race 4 on all three cultivars on a per plant basis but not on a per gram root tissue basis. The control, M. incognita alone, and race 1 alone treatments caused no symptoms. Inoculation with race 1 and M. incognita caused moderate wilt symptoms in 'Acala 44' and 'FM 966' and mild symptoms in 'Pima S-4'. However, race 4 treatment caused severe wilt in 'Pima S-4' and moderate wilt severity in 'Acala 44' and 'FM 966'. The symptom severity of 'Acala 44' and 'FM 966' further increased in the presence of M. incognita. Thus, race 4 is not only capable of causing wilt in the absence of M. incognita but can also interact with the nematode to further increase disease severity. Though control of wilt caused by race 1 can be achieved mainly through breeding for nematode resistance, it will be imperative to incorporate both southern root-knot nematode and race 4 resistance to effectively control the disease should race 4 expand into southern root-knot nematode-infested fields.

Characterization of Current Fusarium oxysporum f. sp. vasinfectum Isolates from Cotton in the San Joaquin Valley of California and Lower Valley El Paso, Texas.

Fusarium oxysporum f. sp. vasinfectum race 4 is a causal agent of Fusarium wilt of cotton (Gossypium spp.). This study aimed to characterize the existing distribution and frequency of current field populations of F. oxysporum f. sp. vasinfectum race 4 genotypes in the San Joaquin Valley (SJV) of California and Lower Valley El Paso, TX and examine representative isolates for aggressiveness during different stages of seedling development. A survey was conducted from 2017 to 2019 across 13 locations in the SJV and one location in El Paso, TX during 2018. From the SJV, isolates identified as the F. oxysporum f. sp. vasinfectum race 4 T genotype were dispersed across the SJV, whereas isolates identified as the F. oxysporum f. sp. vasinfectum race 4 N genotype were most frequently isolated from cotton fields in the northern county of Merced. The F. oxysporum f. sp. vasinfectum race 4 isolates from the Texas location were identified as the MT genotype. A selection of representative isolates was evaluated using three inoculation assays (rolled-towel, F. oxysporum f. sp. vasinfectum-infested oat seed, and root-dip inoculation) to test the isolates' abilities to produce symptoms during seedling stages of cotton development. All isolates tested were capable of producing symptoms on cotton; however, isolate aggressiveness varied within and across inoculation assays. In all assays, higher levels of disease development were observed in the moderately susceptible Pima (Gossypium barbadense L.) cultivars (DP-340 or PHY-830) when compared with the moderately tolerant Upland (G. hirsutum L.) cultivar (FM-2334). However, no correlation was found among the different response variables for the rolled-towel assay when compared with the root-dip and infested oat seed assays. These results suggest that different genes are involved in the resistance response during the early seedling development stage measured in the rolled-towel assay compared with the later seedling development stages measured during the root-dip inoculation and infested oat seed assays, revealing the complexity of the Fusarium wilt disease and host-plant resistance mechanisms.

Detection and Characterization of Fusarium oxysporum f. sp. vasinfectum VCG0114 (Race 4) Isolates of Diverse Geographic Origins.

A highly virulent cotton wilt pathogen, Fusarium oxysporum f. sp. vasinfectum VCG0114 (race 4) was found in West Texas in 2017, after being known in California since 2001. Isolates obtained from wilted plants collected in 2017 from Texas, in 2015 from China, and during 2001 to 2014 from California and isolates from historical collections including the race 4 reference isolate were characterized by soil-infestation pathogenicity assays, DNA sequence analysis, and vegetative compatibility analysis. All obtained F. oxysporum f. sp. vasinfectum isolates belonged to VCG0114. All of these isolates, except one isolate from China, caused disease in a soil-infestation assay without nematodes. Thus, they belong to the nematode-independent pathotype. Texas isolates were significantly more virulent than were isolates from China or California on Gossypium barbadense 'Pima S-7'. Four different genotypes (N, T, MT, and MiT) were identified based on the transposable element Tfo1 insertion into the PHO gene and independent MULE or MITE insertions into the Tfo1 transposon. Some significant differences in virulence were detected among the genotypes in some locations. No differences in pathogenicity were observed between the California and China collection isolates on Pima S-7, and the virulence of the major genotypes was similar on the Gossypium hirsutum cultivar 'Stoneville 474' or the Barbren 713 germplasm line. Simple polymerase chain reaction (PCR) methods were developed to specifically determine and detect the four genotypes within VCG0114. A specific PCR method to detect all VCG0114 isolates was also developed. These methods will facilitate the timely identification of infested fields and seed lots and the elucidation of evolutionary relationships among the isolates. This should help to closely monitor the movement of the pathogen and reduce dissemination of these devastating pathogens.

Detection and Genotyping of Fov4 (Race 4, VCG0114), the Fusarium Wilt Pathogen of Cotton.

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), is an important disease of cotton. More than 14 different genotypes as determined by VCG and sequence analyses are known to occur in the United States. Fov4 (race 4, VCG0114), originally found in India, was first detected in the United States in 2001 in California and recently in 2017 and 2019 in Texas and New Mexico, respectively. Four sub-genotypes of Fov4 have been identified, with Fov4 N, T, and MiT genotypes occurring in California, and Fov4 T and MT genotypes occurring in Texas. Unlike other genotypes of Fov in the United States, Fov4 does not require the presence of root-knot nematodes (Meloidogyne incognita) to cause severe wilt in cotton and is a major concern to US cotton growers. Fov4 can be spread through a variety of mechanisms including through infected seed. Once a field is infested, the fungus becomes endemic since there are no economically viable means to eradicate the pathogen from infested fields. Therefore, a rapid and accurate detection method is essential for early identification of infested fields and seed lots to prevent further spread of Fov4. This chapter describes multiplex and singleplex PCR diagnostics for detection of Fov4, and for detection and genotyping N, T, MiT, and MT genotypes of Fov4 from wilted cotton plants.

Differences in Active Defense Responses of Two Gossypium barbadense L. Cultivars Resistant to Fusarium oxysporum f. sp. vasinfectum Race 4.

A highly virulent race 4 genotype of Fusarium oxysporum f. sp. vasinfectum (Fov) was identified for the first time in the western hemisphere in 2002 in cotton fields in the San Joaquin Valley of California. The Gossypium barbadense L. cotton cultivars 'Seabrook Sea Island 12B2' ('SBSI') and 'Pima S-6' are resistant to Fov race 4. Active defense responses were quantitated by monitoring the accumulation of antimicrobial terpenoids (i.e., phytoalexins) in inoculated stem stele tissue in these cultivars. The increase in the concentration of the most toxic phytoalexins was statistically faster after 24 h in 'SBSI' compared to 'Pima S-6'. The sesquiterpenoid hemigossylic acid lactone, which was observed for the first time in nature, also accumulated in diseased plants. Neither hemigossylic acid lactone nor the disesquiterpenoids gossypol, gossypol-6-methyl ether, and gossypol-6,6'-dimethyl ether showed toxicity to Fov. Segregation of F2 progeny from 'SBSI' × 'Pima S-6' crosses gave a few highly susceptible plants and a few highly resistant plants, indicating separate genes for resistance in the two cultivars.

Detoxification of Fusaric Acid by the Soil Microbe Mucor rouxii.

Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114), which causes root rot and wilt of cotton (Gossypium hirsutum and G. barbadense), has been identified recently for the first time in the western hemisphere in certain fields in the San Joaquin Valley of California. This pathotype produces copious quantities of the plant toxin fusaric acid (5-butyl-2-pyridinecarboxylic acid) compared to other isolates of F. oxysporum f. sp. vasinfectum (Fov) that are indigenous to the United States. Fusaric acid is toxic to cotton plants and may help the pathogen compete with other microbes in the soil. We found that a laboratory strain of the fungus Mucor rouxii converts fusaric acid into a newly identified compound, 8-hydroxyfusaric acid. The latter compound is significantly less phytotoxic to cotton than the parent compound. On the basis of bioassays of hydroxylated analogues of fusaric acid, hydroxylation of the butyl side chain of fusaric acid may affect a general detoxification of fusaric acid. Genes that control this hydroxylation may be useful in developing biocontrol agents to manage Fov.