Vacuolar Processing Enzymes Modulating Susceptibility Response to Fusarium oxysporum f. sp. cubense Tropical Race 4 Infections in Banana

Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) is a destructive necrotrophic fungal pathogen afflicting global banana production. Infection process involves the activation of programmed cell death (PCD). In this study, seven Musa acuminata vacuolar processing enzyme (MaVPE1–MaVPE7) genes associated with PCD were successfully identified. Phylogenetic analysis and tissue-specific expression categorized these MaVPEs into the seed and vegetative types. FocTR4 infection induced the majority of MaVPE expressions in the susceptible cultivar “Berangan” as compared to the resistant cultivar “Jari Buaya.” Consistently, upon FocTR4 infection, high caspase-1 activity was detected in the susceptible cultivar, while low level of caspase-1 activity was recorded in the resistant cultivar. Furthermore, inhibition of MaVPE activities via caspase-1 inhibitor in the susceptible cultivar reduced tonoplast rupture, decreased lesion formation, and enhanced stress tolerance against FocTR4 infection. Additionally, the Arabidopsis VPE-null mutant exhibited higher tolerance to FocTR4 infection, indicated by reduced sporulation rate, low levels of H2O2 content, and high levels of cell viability. Comparative proteomic profiling analysis revealed increase in the abundance of cysteine proteinase in the inoculated susceptible cultivar, as opposed to cysteine proteinase inhibitors in the resistant cultivar. In conclusion, the increase in vacuolar processing enzyme (VPE)-mediated PCD played a crucial role in modulating susceptibility response during compatible interaction, which facilitated FocTR4 colonization in the host.

DNA-based quantification of Fusarium oxysporum f. sp. vasinfectum in environmental soils to describe spatial variation in inoculum density

Fusarium wilt of cotton, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (FOV), occurs in regions of the United States where cotton (Gossypium spp.) is grown. Race 4 of this pathogen (FOV4) is especially aggressive and does not require the co-occurrence of the root knot nematode (Meloidogyne incognita) to infect cotton. Its sudden appearance in far-west Texas in 2016 after many years of being restricted to California is of great concern, as is the threat of its continued spread through the cotton-producing regions of the United States. The aim of this research was to analyze the spatial variability of FOV4 inoculum density in the location where FOV4 is locally emerging, using quantitative and droplet digital polymerase chain reaction (qPCR and ddPCR) methods. Soil samples collected from a field with known FOV4 incidence in Fabens, Texas were analyzed. Appreciable variation in inoculum density was found to occur at spatial scales smaller than the size of plots involved in cultivar trial research, and was spatially autocorrelated (Moran’s I, Z = 17.73, p < 0.0001). These findings indicate that for cultivar trials, accounting for the spatial distribution of inoculum either by directly quantifying it or through the use of densely-distributed “calibration checks” is important to the interpretation of results.

First report of Fusarium oxysporum f. sp. cubense Tropical Race 4, causing Fusarium wilt in Cavendish bananas in Peru

Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), the causal agent of Fusarium wilt of banana (FWB), is currently the major threat to the banana industry worldwide (Dita et al. 2018). Restricted to South Asia for more than 20 years, Foc TR4 has spread in the last years to the Middle East, Mozambique, and Colombia (García-Bastidas et al. 2019; https://pestdisplace.org/embed/news/map/disease/11). The incursion of Foc TR4 in Colombia increased awareness and prevention efforts across Latin American and the Caribbean (LAC). However, new Foc TR4 outbreaks in LAC countries were considered a matter of time. In April 2021, banana (Musa spp., Cavendish, AAA) plants (30% of incidence) showing typical symptoms of FWB, such as leave yellowing, wilting and vascular discoloration were observed in one farm (about 1 ha) located in Querecotillo, Peru (4°43’54.84”S 80°33’45.00”W). Mycological analyses of samples (pseudostem strands) collected from 10 symptomatic plants were performed as described by Dita et al. (2010). These analyses revealed a continuous presence of fungal colonies identified as Fusarium oxysporum species complex. Molecular diagnostics targeting two different genome regions (Dita et al 2010; Li et al. 2013) identified nine of these isolates as Foc TR4. These results were further confirmed by qPCR analyses using the commercial Clear Detections TR4 kit. The genome of four single-spore isolates (PerS1, PerS2, PerS3 and PerS4) was sequenced using the Illumina platform (MiSeq Kit, 2x151 bp Paired-End). The strain PerS4 was also sequenced using Oxford Nanopore (FLOW-MIN111; R10.3 chemistry) as described by Lopez-Alvarez et al., (2020). The generated draft assembly yielded 533 contigs for a size of 47 Mbp (BioProject: PRJNA755905), which is comparable with sizes of previously reported Foc TR4 strains (Asai et al. 2019; García-Bastidas et al. 2019; Maymon et al. 2020; Warmington et al. 2019; Zheng et al. 2018). The sequence assembly showed high contiguity (94.9%) and high similarity (95.48%) with the high-quality genome sequence of the Foc TR4 isolate ‘UK0001’ (Warmington et al. 2019). Further analyses to identify the presence/absence of full sequences for the putative effector genes (Secreted In Xylem - SIX) and their allelic copies, also revealed that the SIX genes profile of the strains isolated from Querecotillo matched with previously reported Foc TR4 isolates (Czislowski et al. 2017). Pathogenicity tests with three isolates and water controls were performed as described by Dita et al. (2010), using five Cavendish plantlets per treatment. Four weeks after the inoculation typical external and internal symptoms of FWB were observed only in the inoculated plants. Fungal isolates recovered from inoculated plants tested positive for Foc TR4 when analyzed with PCR diagnostics as mentioned above. No fungal isolates were recovered from water-control plants which did not show any symptoms. Altogether, our results confirm the first incursion of Foc TR4 in Peru. Currently, Foc TR4 has the phytosanitary status of a present pest with restricted distribution in Peru and it is under official control of the National Plant Protection Organization – SENASA. Reinforced prevention and quarantine measures, disease monitoring and capacity building to detect, contain and manage eventual new outbreaks of Foc TR4 are strongly encouraged across LAC banana producing countries, especially for those bordering Peru with larger banana plantations, such as Ecuador and Brazil.

Intraspecies multiple chromosomal variations including rare tandem fusion in the Russian Far Eastern endemic evoron vole Alexandromys evoronensis (Rodentia, Arvicolinae)

The vole Alexandromys evoronensis (Kovalskaya et Sokolov, 1980) with its two chromosomal races, “Evoron” (2n = 38–41, NF = 54–59) and “Argi” (2n = 34, 36, 37, NF = 51–56) is the endemic vole found in the Russian Far East. For the “Argi” chromosomal race, individuals from two isolated populations in mountain regions were investigated here for the first time using GTG-, GTC-, NOR methods. In the area under study, 8 new karyotype variants have been registered. The karyotype with 2n = 34 has a rare tandem fusion of three autosomes: two biarmed (Mev6 and Mev7) and one acrocentric (Mev14) to form a large biarmed chromosome (Mev6/7/14), all of which reveal a heterozygous state. For A. evoronensis, the variation in the number of chromosomes exceeded the known estimate of 2n = 34, 36 and amounted to 2n = 34, 36, 38–41. The combination of all the variations of chromosomes for the species made it possible to describe 20 variants of the A. evoronensis karyotype, with 11 chromosomes being involved in multiple structural rearrangements. In the “Evoron” chromosomal race 4 chromosomes (Mev1, Mev4, Mev17, and Mev18) and in the “Argi” chromosomal race 9 chromosomes (Mev6, Mev7, Mev14, Mev13, Mev11, Mev15, Mev17, Mev18, and Mev19) were observed. Tandem and Robertsonian rearrangements (Mev17/18 and Mev17.18) were revealed in both chromosomal races “Evoron” and “Argi”.

Acquisition and Transmission of Fusarium oxysporum f. sp. vasinfectum VCG 0114 (Race 4) by Stink Bugs

Fusarium oxysporum f. sp. vasinfectum VCG 0114 (race 4; i.e., FOV4) is an emerging pathogen that causes severe root rot and wilt of cotton. FOV4 is seed-borne, but the mode of seed invasion is uncertain. In an initial study, seeds in bolls that were puncture inoculated with FOV4 conidia when they were 25- or 30-days old became infected but remained viable. Because stink bugs can ingest and introduce bacterial and yeast pathogens into cotton bolls, we hypothesized that stink bugs may ingest and transmit FOV4. Southern green stink bugs and brown stink bugs were exposed to potato dextrose agar cultures of FOV4 and subsequently caged with cotton bolls to assess transmission potential. Both species fed on the cultures and acquired FOV4, and brown stink bugs transmitted FOV4 to cotton bolls. Thus, management of FOV4 may require management of stink bugs to mitigate the spread of the disease in cotton.

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.