Source: TEXAS A&M UNIVERSITY submitted to NRP
ENHANCING RESISTANCE IN UPLAND COTTON BY DISSECTING THE DISEASE MECHANISM OF FUSARIUM OXYSPORUM F. SP. VASINFECTUM RACE 4
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1019783
Grant No.
2019-67028-29949
Cumulative Award Amt.
$294,000.00
Proposal No.
2018-09106
Multistate No.
(N/A)
Project Start Date
Jul 15, 2019
Project End Date
Jul 14, 2023
Grant Year
2019
Program Code
[A1701]- Critical Agricultural Research and Extension: CARE
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Plant Pathology & Microbiology
Non Technical Summary
A highly virulent strain of Fusarium wilt of cotton caused substantial losses in California cotton production. The strain was then detected in 2017 in a Pima cotton field east of El Paso, Texas. The overal goal is to understand the fungal infection process and provide essential genomic resources to cotton researchers/breeders.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011710116034%
2011710108133%
2011710108033%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1710 - Upland cotton;

Field Of Science
1160 - Pathology; 1081 - Breeding; 1080 - Genetics;
Goals / Objectives
1. Understanding the infection process and genetic identity of Fov4. To develop effective disease controls and containment strategies, we aim to monitor the fungal attachment, penetration, colonization and establishment by live-cell imaging through the course of infection period using green fluorescent protein (GFP)-tagged Fov4 isolates. To reveal the genetic nature of Fov4 isolates, we will perform whole-genome sequencing using Oxford Nanopore MinION portable device. 2. Germplasm screens of cotton resistance to Fov4. To address a pressing need on developing upland cotton with Fov4 resistance, a coordinated effort of large-scale field screens in determining the genetic variation of existing upland breeding lines to Fov4 infections is ongoing in the fields of El Paso, TX. More than 2,000 lines represent the breadth of diversity, as well as a representative sample of upland accessions, specifically those used successfully in introgression of adapted genotypes. 3. Characterization of candidate Fov4 resistance genes introgressed into upland lines and Pima S-6 (G. barbadense). The resistance source in upland cotton germplasm is likely introgressed from G. barbadense from sources such as Pima S-6, which carries a major dominant resistance gene originally mapped on chromosome (Chr.) 14 and later also on Chr. 17, a translocation region of the cotton genome with possible gene duplication with allelic dosage effect for Fov4 resistance/tolerance, as well as, other minor quantitative trait loci contribution from other chromosomes. We will examine the involvement of candidate genes in these regions for their resistance against Fov4.
Project Methods
Objective 1. Fusarium wilt disease of cotton (Gossypium spp.), caused by soil-borne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (Fov), has been a continuing problem causing cotton losses worldwide. This pathogen is particularly difficult to control in cotton as the hyphae reside in the woody vascular tissues and is thus protected from fungicides with overwintering structures that can survive in soils forever. Fov race 4 (Fov4), identified in the U.S. in 2003, was known to be distributed only in California previously, but was identified in Texas in 2017. Resistance to Fov4 was originally identified in commercial Pima cotton (G. barbadense L.), 'Phytogen 800', and originated-pool germplasm, 'Pima S-6'. However, so far, resistance in upland cotton (G. hirsutum L.) has not been identified and commercial varieties are not available. Thus, there is a critical need to further understand the disease mechanism and cotton. We will develop effective disease controls and containment strategies, we aim to monitor the fungal attachment, penetration, colonization and establishment by live-cell imaging through the course of infection period using green fluorescent protein (GFP)-tagged Fov4 isolates. To reveal the genetic nature of Fov4 isolates, we will perform whole-genome sequencing using Oxford Nanopore MinION portable device.Objective 2. To address a pressing need on developing upland cotton with Fov4 resistance, a coordinated effort of large-scale field screens in determining the genetic variation of existing upland breeding lines to Fov4 infections is ongoing in the fields of El Paso, TX. More than 2,000 lines represent the breadth of diversity, as well as a representative sample of upland accessions, specifically those used successfully in introgression of adapted genotypes.Objective 3. Fusarium wilt disease of cotton, caused by soil-borne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (Fov), can be a serious disease in most cotton producing countries. This pathogen is particularly difficult to control in cotton as the hyphae reside in the woody vascular tissues and is thus protected from fungicides with overwintering structures that can survive in soils forever. The fungus infects at any stage of cotton development, often resulting in seedling death similar to damping off, growth stunting, ultimately wilt and death. We will study Characterization of candidate Fov4 resistance genes introgressed into upland lines and Pima S-6 (G. barbadense). The resistance source in upland cotton germplasm is likely introgressed from G. barbadense from sources such as Pima S-6, which carries a major dominant resistance gene originally mapped on chromosome (Chr.) 14 and later also on Chr. 17, a translocation region of the cotton genome with possible gene duplication with allelic dosage effect for Fov4 resistance/tolerance, as well as, other minor quantitative trait loci contribution from other chromosomes. We will examine the involvement of candidate genes in these regions for their resistance against Fov4. We will learn whether by breeding or genetic engineering techniques, is needed to mitigate against the destructive potential of Fusarium wilt. However, both of these techniques require a targeted approach which builds upon a comprehensive knowledge of the cotton-Fov4 interactions, including how the pathogen attacks the plants and how the plants react with defense responses.

Progress 07/15/19 to 06/20/23

Outputs
Target Audience:The cotton scientific community, the general scientific community, and agricultural-related sectors Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Catherine Danmaigona Clement, aPh.D. student, graduated in the spring of 2023 John Christian Hitzelberger, aPh.D. student, graduated in the Fall of2022 Jennifer Chagoya, a Ph.D. student at Texas Tech University has been working on Fov4 related project. Alexis Schultz, an M.S student, graduated in the spring of 2023 How have the results been disseminated to communities of interest?Findings are disseminated primarily through peer-reviewed journal articles, presentations, conferences, and symposiums. We also collaborate with cotton field extension specialists. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Development of FOV4 in resistant and susceptible germplasm The plant breeders at Texas A&M and plant pathologists have been working together since 2018 at a FOV4 infested field in Fabens, TX. Each year (2018-2022), breeding lines, germplasm from designed crosses, and various commercial checks have been rated for plant mortality (percentage of plants that died after initial emergence) and root vascular discoloration, which are the key indicators of FOV4 damage. The commercial checks include susceptible Pima varieties (DP 357, PHY 830, DP 744), resistant Pima varieties (PHY 881 RF, PHY 841 RF, DP 359 RF), susceptible Upland variety (PHY 480 W3FE), and a partially resistant/tolerant Upland variety (FM 2334GLT). In general, FM 2334GLT can be characterized by low plant mortality and relatively low root vascular staining. FOV4 resistant Pima varieties generally have slightly higher plant mortality due to FOV4, but almost no root vascular discoloration. The primary target of the Texas A&M breeding programs are to develop resistant or partially resistant FOV4 Upland cotton cultivars, with the goal of surpassing the tolerant response of FM 2334GLT. In 2019, most of the efforts were still directed at screening lines within the Texas A&M breeding programs, so one large test was conducted with approximately 300 lines and 3 replications per line. In terms of mortality, 15% of the Texas A&M lines had equal or lower mortality than FM 2334GLT, and the mortality was also lower (better) than the resistant Pima check. For root vascular discoloration, the staining was far more advanced in the December root ratings than the July root ratings, and statistically there were stronger separations between entries. A decision was made to go forward only with the late season root ratings. There were 15 entries that had lower root discoloration ratings than FM 2334GLT, and 6 lines with lower root ratings than the resistant Pima check. There were 7 breeding lines that were considered high performing in both mortality and root vascular discoloration. From 2020 - 2022, the emphasis shifted more to testing the progeny from crosses and subsequent generations. For example, in 2020, there were 3 different types of tests (EP1, EP2, EP3) that were conducted at the Fabens FOV4 field. These tests contained 174, 42, and 185 experimental lines, respectively. In the first test (EP1), 15% of the germplasm had numerically lower plant mortality than FM 2334GLT. In the second test, which included both mortality and vascular root damage ratings, 57% of the entries had lower mortality ratings and 12% of the entries had lower root discoloration ratings than FM 2334GLT. In the third test, 6% and 1% of the entries had lower mortality or root discoloration ratings, respectively than FM 2334GLT. In 2021, there were two tests conducted, EP3 with 24 entries and EP5 with 245 entries. In EP3 14% of the entries had lower mortality and 75% of the entries had less root vascular discoloration than FM 2334GLT. In EP5, 22% of the entries had lower mortality (no root ratings) than FM 2334GLT. In 2022, an F2 population was tested (total of 24 entries), and FM 2334GLT had an exceptionally high mortality rate. All the F2 progeny had lower mortality than FM 2334GLT, while 12 entries had lower morality than the resistant Pima check. Progress is being made with the development of Upland germplasm that demonstrate relatively low mortality and root vascular discoloration to FOV4. Following initial 2018 screening of 2,000 cotton lines provided by four Texas A&M cotton breeding/genetics programs, seed was increased from a subsample of ~400 lines for additional screening, data collection, and cultivar development. Screening in 2019 was conducted in an area of the field deemed most densely and uniformly infested with Fov4. Parents with potential for breeding Fov4 resistance were identified after two years of field screening, and first filial generation seed from over 50 cross-pollinations was increased. Segregating second filial (F2) generation seed was evaluated in the field in 2020, and additional crosses made after data collection from 2020 screening. Third filial generation progeny rows and additional F2 populations were evaluated in 2022. In 2022, ten near-homozygous, uniform F4 lines are being tested in a 28-entry small-plot replicated trial along with more advanced material from cotton breeding programs at New Mexico State University and USDA-ARS, and four commercial check varieties with varying response to Fov4. Initial indications are candidate cultivars show improved agronomic and fiber characteristics compared to current known sources of resistance. Unexpected genomic alterations within Fusarium oxysporum f. sp. vasinfectum isolates reveal the evolution and pathogenicity of F. oxysporum and its accessory chromosomes. Fusarium oxysporum is a cross-kingdom pathogen that causes notorious diseases with the most diverse host range, including plants, animals, and humans. Different isolates in F. oxysporum species complex (FOSC) were defined as formae speciales based on their host specificity. F. oxysporum f. sp. vasinfectum (Fov) causes Fusarium wilt in cotton, which could lead to up to 80% yield loss. FOSC possesses complex genome structures with a set of core chromosomes and diverse small accessory chromosomes that are assumed to associate with virulence and host specificity. It remains unknown the genetic diversity and evolution of different isolates within a formae speciales. We sequenced and assembled high-quality genomes of two classical Fov races and two newly evolved isolates in a naturally infected field in Texas, USA. Comparative genomic analysis revealed that Fov isolates share 11 core chromosomes with other FOSC isolates that infect both plants and humans, and a group of unique accessory chromosomes. Surprisingly, although the core chromosomes are highly conserved, the accessory chromosomes are extremely divergent within Fov isolates, with the same degree of divergence among different formae speciales. Consistently, phylogenetic analysis indicates the independent evolution of different Fov races within FOSC, and different Fov isolates could infect multiple hosts. Extensive microsynteny was observed among accessory chromosomes within the same or different FOSC isolates, suggesting the existence of a shared ancestral accessory chromosome in FOSC. Genome sequencing of Texas Fov field isolates differing in virulence further revealed the rapid genome structure and sequence changes of accessory chromosomes within the Fov race. The increased pathogenicity of some newly evolved Fov isolates is associated with the increased copy number of putative effector genes in the accessory chromosomes. Together, our data reveal that different isolates in the same formae speciales of F. oxysporum could have evolved independently, and the accessory chromosomes in FOSC might have a common ancestor of evolution. Our analysis also suggests that massive whole accessory chromosome duplications, intrachromosomal segmental duplications, chromosome rearrangements, gene losses and gains, and transposable element expansion in different FOSC isolates drive pathogen aggressiveness and host adaptation. A manuscript about this work is in preparation for submission.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhang, J., A. Abdelraheem, Y. Zhu, T. A. Wheeler, J. K. Dever, R. L. Nichols, and T. Wedegaertner. 2021. Importance of temperature in evaluating cotton for resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum race 4. Crop Science. 61(3): 1783-1796. https://doi.org/10.1002/csc2.20446
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhu, Y., A. Abdelraheen, T. A. Wheeler, J. K. Dever, T. Wedegaertner, K. Hake. And J. Zhang. 2021. Interactions between cotton genotypes and Fusarium wilt race 4 isolates from Texas and resistance evaluation in cotton. Crop Science. 61(3): 1809-1825. https://doi.org/10.1002/csc2.20469
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Abdelraheem, A., Y. Zhu, J. K. Dever, T. A. Wheeler, T. Wedegaertner, K. Hake, and J. Zhang. 2021. Diallel analysis of resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in American Pima cotton, Gossypium barbadense L Crop Science. 61(6):4000-4011. https://doi.org/10.1002/csc2.20606
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhu, Y., A. Abdelraheem, P. Cooke, T. A. Wheeler, J. K. Dever, T. Wedegaertner, K. Hake, and J. Zhang. 2021. Comparative analysis of infection process in Pima cotton differing in resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum race 4. Phytopathology.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Dever, J. K., C. M. Kelly, and V. A. Morgan. Registration of cotton germplasm line CA 4008. 2022. Journal of Plant Registrations. 16:87-93. https://doi.org/10.1002/plr2.20175.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhang, J., Y. Zhu, A. Abdelraheem, H. D. Elkins-Arce, J. K. Dever, T. A. Wheeler, T. Isakeit. K. Hake, and T. Wedegaertner. 2021. Use of a latin square design to assess experimental errors in field evaluation of cotton for resistance to Fusarium wilt race 4. Crop Science. https://doi.org/10.1002/csc2.20673
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Zhang, J., A. Abdelraheem, J. Ma, Y. Zhu, J. K. Dever, T. A. Wheeler, K. Hake, T. Wedegaertner, and J. Yu. 2022. Mapping of dynamic QTLs for resistance to Fusarium wilt (Fusarium osysporum f. sp. vasinfectum) race 4 in a backcross inbred population of upland cotton. Molecular Genetics and Genomics. https://doi.org/10.1007/s00438-021-01846-2.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Zhang, J., A. Abdelraheem, Y. Zhu, H. Elkins-Arce, J. K. Dever, D. Whitelock, K. Hake, T. Wedegaertner, and T. A. Wheeler. 2022. Studies of evaluation methods for resistance to Fusarium wilt race 4 (Fusarium oxysporum f. sp. vasinfectum) in cotton: effects of cultivar, planting date, and inoculum density on disease progression. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2022.900131
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Zhang, J., Y. Zhu, H. Elkins-Arce, T. A. Wheeler, J. K. Dever, D. Whitelock, K. Hake, and T. Wedegaertner. 2022. Studies of evaluation parameter for resistance to Fusarium wilt in cotton caused by Fusarium wilt race 4 (Fusarium oxysporum f. sp. vasinfectum). Crop Science. 16(3): 1115-1132. https://doi.org/10.1002/csc2.20744
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Zhu, Y., H. Elkins-Arce, T. A. Wheeler, J. K. Dever, D. Whitelock, K Hake, T. Wedegaertner, and J. Zhang. 2022. Effect of growth stage, cultivar, and root wounding on disease development in cotton caused by Fusarium wilt race 4 (Fusarium oxysporum f. sp. vasinfectum). Crop Science. https://doi.org/10.1002/csc2.20839.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Lakey, C., T. A. Wheeler, L. Shan. J. K. Dever, and S. Hague. 2020. Improving cotton resistance to FOV race 4. Beltwide Cotton Research Conference. January 9, Austin, Texas.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Abdelraheen, A., Y. Zhu, T. A. Wheeler, J. K. Dever, J. Yu, Y. Yuan, Y. Shi, Y., J. Ma, T. Wedegaertner, and J. Zhang. 2021. Genetic mapping for resistance to fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in two introgressed populations of upland cotton (Gossypium hirsutum). Proceedings of the Beltwide Cotton Research Conference. January 6, Virtual.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Ulloa, M. R. Hutmacher, P. A. Roberts, M. Ellis, T. L. Frigulti, J. K. Dever, T. A. Wheeler, S. Hague, J. Zhang, S. Sonogo, P. Payton, and R. L. Nichols. 2021. Resistance/tolerance to Fusarium wilt race 4 (FOV4) in Upland cotton (Gossypium hirsutum L.) for germplasm public release. Beltwide Cotton Research Conference. National Cotton Council of America.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Jung. J., J. Landivar, A. Chang, J. K. Dever, J. Zwonitzer, A. Maeda, and M. Maeda. 2021. Unmanned Aerial System (UAS)-based plant stand monitoring for Fov4 resistance evaluation. Beltwide Cotton Research Conference. January 6, Virtua
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Danmaigona Clement, C., Z. Liu, L. Zhang, E-G. No, S. Hague, T. A. Wheeler, P. He, L. Shan, J. K. Dever, and F. A. Ortiz. 2022. Dissecting the genome structure of Fusarium oxysporum f. sp. vasinfectum race 4 causing devastating losses to cotton [Abstract]. ASA, CSSA, SSSA International Annual Meeting, Salt Lake City, UT https://scisoc.confex.com/scisoc/2021am/meetingapp.cgi/Paper/135233.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Schultz, A., S. Hague, J. K. Dever, T. A. Wheeler, T. Isakiet, and C. Lakey. 2021 Improvement of host plant resistance in cotton to FOV4 [Abstract]. ASA, CSSA, SSSA International Annual Meeting, Salt Lake City, UT. https://scisoc.confex.com/scisoc/2021am/meetingapp.cgi/Paper/135780
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Danmaigona Clement, C., Z. Liu, L. Zhang, E-G. No, S. Hague, T. A. Wheeler, P. He, L. Shan, and J. K. Dever. 2021. Genomic plasticity and virulence diversity of Fusarium oxysporum f. sp. vasinfectim Race4 from Cotton Fields [Abstract]. ASA, CSSA, SSSA International Annual Meeting, Salt Lake City, UT. https://scisoc.confex.com/scisoc/2021am/meetingapp.cgi/Paper/135155
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Clement, C. D., Z. Liu, E-G No, L. Zhang, S Hague, T. A. Wheeler, P. He, J. K. Dever, and L. Shan. 2022. Genome plasticity of Fusarium oxysporum f. sp. vasinfectum from cotton fields. Beltwide Cotton Research Conference. January 5, San Antonio, Texas.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Shultz, A., S. Hague, J. K. Dever, T. A. Wheeler, T. Isakeit, and C. Lakey. 2022. Improvement of host plant resistance in cotton to FOV4 [Abstract]. ASA, CSSA. SSSA International Annual Meeting, Baltimore, MD. https://scisoc.confex.com/scisoc/2022am/meetingapp.cgi/Paper/143189


Progress 07/15/21 to 07/14/22

Outputs
Target Audience:The cotton scientific community, the general scientific community, and agricultural-related sectors Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has made available several avenues for training postdoctoral fellows, graduate students, and undergraduates. How have the results been disseminated to communities of interest?Findings are disseminated primarily through peer-reviewed journal articles, presentations, conferences, and symposiums. We also collaborate with cotton field extension specialists. What do you plan to do during the next reporting period to accomplish the goals?Our research will continue as proposed advancing towards our goals in the original proposal. We are working towards timely publication of our research findings to the scientific community, validating resistance gene candidates, training Postdocs, graduate, and undergraduate students.

Impacts
What was accomplished under these goals? Origin of lineage-specific chromosomes in Fusarium oxysporum Although LS chromosomes in Fov1 and Fov4 are distinct and unique to each race, Fov4 LS chromosomes are more extensive. 45% of Fov1 LS genes have at least one orthologous gene in Fov4. Importantly, among these orthologs, 6.5% of genes in Fov1 are duplicated twice, 10.7% three times, and 9.9% four times or more in different Fov4 LS chromosomes. For example, a 30 kb fragment of Fov1 LS Chr. 6 has duplications in Fov4 LS, two in Chr. 3, and two in Chr. 6. The data suggests that Fov1 and Fov4 LS chromosomes could be derived from the same ancestral chromosomes, and there could be whole chromosome duplications, intrachromosomal segmental duplications (such as Chr.6), chromosome rearrangements, gene losses, gene gains, and TE expansion in Fov4. This is also consistent with the field observation in which Fov1 is a more ancient race and is the most widely isolated race on infected cotton plants. Interestingly, 42 Fov1 LS genes at least one orthologous gene in Fov4 Chr. 12a and 12b of Fov4. For example, the 1kb fragment in Fov1 Chr.6 has two duplicates in Fov4 Chr. 12a and one duplicate in Chr. 12b, suggesting that these two chromosomes underwent chromosome fusion with LS chromosomes after the breakage and explaining the high TE contents in these two chromosomes. A similar event could happen for Fov4 Chr. 1, which is significantly larger than Fov1 and Fol counterparts. Macro synteny plots of Fov1 LS genes and Fov4 LS, Chr12a and Chr12b genes are represented in Fig. 5D. Gene losses (Fov1 LS genes with no ortholog in Fov4) of 55% and 10.4% are observed in the LS and core chromosomes of Fov4 respectively, depicting that LS chromosomes are more vulnerable to gene losses. Thus, the increased aggressiveness of Fov4 is likely due to LS chromosome duplications and rearrangements, and fusion with core chromosomes, and instability resulting in an increased copy number of pathogenicity factors. We propose that the origin of LS chromosomes in Fov4 is different from the proposed horizontal acquisition from another Fusarium species for Fol. The high content of TEs in LS chromosomes is likely the driving force of these chromosomal structural changes. Next, we reasoned that Fusarium oxysporum f. sp. radicis-cucumerinum (Forc16) reported to possess the mobile pathogenicity accessory chromosome chrRC 1 might be more ancient than Fov1 and could be the potential originator of these duplicated LS genes mostly because it possessed only one LS chromosome. Nonpathogenic strains became pathogenic on cucurbit species after acquiring this chromosome through horizontal chromosome transfer1. To verify this, we carried out extensive syntenic gene analysis between Forc16 (reference) and Fov1 and we observed syntenic genes and duplicates of Forc16 LS genes and some gene in Chr. 11 in Fov1 LS chromosomes (3, 6). Similar observations were made between Forc16 (reference) and Fol comparison with syntenic duplicate genes of Forc16 chrRC, Chr.11 and Chr.12 present in the Fol LS chromosomes (3, 6, 14, 15) and some parts of the Chr1. and Chr2. Previously reported to be LS. This resulting data suggested that Fov1 and Fol LS might be derived from the same ancient ancestral chromosome, possibly Forc16 chrRC followed by massive duplications. Several gene duplications and fusion events in core and LS chromosomes are an overlooked cause of the evolution of LS chromosomes within the FOSC. We observe that some core chromosome segment duplication of these genes could give rise to new chromosomes probably driven by fusion events and high enrichment of transposons, as well as multiple duplicates of genes in the LS chromosomes of previous ancestors. This brings the origin of LS chromosomes into a new perspective. ??WGD Paralogous effector genes are responsible for extra virulent race4 isolates Like previously mentioned, race4 isolates Fov4 (virulent isolate), TX_17-3 (virulent isolate) and TX_18-24(extra-virulent isolate) could not be distinguished based on genomic categorization and genome-wide SNPs distribution. They also show a 99.9% average nucleotide similarity with one another thus the extra-virulence of TX_18-24 arises from other pathogenic determinants. As previously stated, many genes in Fov1 LS chromosomes, especially putative effectors, are multifold in Fov4, likewise we also observed Fov4 LS chromosomes, especially putative effectors, are multifold in TX_18-24. All putative effectors in Fov4 LS chromosomes as well as their duplicates in Fov4 and TX_18-24 were obtained. Overall, TX_18-24 recorded more duplicate effector genes than Fov4. Notably these multifold effector copies do not have known functional annotations. Whether these paralogous effector genes with more copies in TX_18-24 will have an overcompensation or increased virulence when deleted remains unknown. These putative paralogous effectors with more copies in an extra virulent strain TX_18-24 compared to a virulent strain and the historic race4 from California Fov4 could potentially contribute to the aggressiveness of TX_18-24. To test this hypothesis, we overexpressed these effectors, in Fov4, and test whether the transformants will increase the virulence. These effectors were cloned into PDL2 vectors and expressed under RP27 (ribosomal protein 27) promoter which is a strong constitutive promoter from M. oryzae and commonly used in filamentous phytopathogenic fungi2. The resultant vector was used to transform Fov4 by a polyethylene glycol (PEG)-mediated protoplast transformation method to generate transformants expressing corresponding effectors 2,3. We selected 4 candidate effector genes with more copies in TX_18-24 to overexpress in Fov4 designated FOVR1824_19288. FOVR1824_18251, FOVR1824_18280 and FOVR1824_19617. Two colonies for each effector gene were obtained after overexpression in Fov4. The overexpression Fov4 strains were then used for pathogenicity test in a susceptible Upland cotton cultivar PHY 499WRF along with a water control and TX_18-24 to ascertain their virulence. Wilting phenotypes were observed and obtained on an average of 6 plants at 14 and 21-days post inoculation with Fov spores. Anova table reveals significant differences in the virulence of these isolates with all the overexpression colonies (except one colony of FOVR1824_18280) causing significantly more wilting at 14 and 21 days post inoculation compared to Fov4. This leads us to conclude that the number of copies of effector genes a race4 isolate possesses determines the level of virulence of the isolate explain the increased level of virulence diversity of race4 isolates obtained from the field. ?Objective 3 We obtain 7.3Tb of raw data for 384 upland cotton lines sequenced at an average depth of 12x. Raw reads were trimmed with cutadapt to remove standard adaptor sequences from illumina sequencing. Trimmed reads were aligned to reference genome Gossypium hirsutum (AD1) 'TM-1' genome UTX_v2.14 using BWA. Mapped reads were sorted with samtools5 and duplicates were marked using picard. Marked duplicate bam files were used for variant calling with both GATK and maintaining the GATK best practices. Total SNPs obtained for 362 samples before and after hard filtering were 612,023 and 552,880 respectively. Total Indels obtained for 362 samples before and after hard filtering were 158,976 and 155,927 respectively. The population level SNPs are currently being used for GWAS analysis to map the region of resistance and obtain candidate genes and segregating haplotypes. Candidate genes and positive haplotypes resulting from the association studies will be further validated to ascertain their function and role in Fov resistance using Virus-Induced Gene Silencing technique (VIGs) for cotton optimized in our lab.

Publications


    Progress 07/15/20 to 07/14/21

    Outputs
    Target Audience:Cotton scientific community, general scientific community and agricultural-related sectors Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has made available several avenues for training postdoctoral fellows, graduate students, rotation students and undergraduates. How have the results been disseminated to communities of interest?Findings are disseminated primarily through peer-reviewed journal articles, presentations, conferences, and symposiums. We presented our work in professional meetings such as ASPB (American Society of Plant Biology Annual Meeting), ASA CSSA & SSSA (American Society of Agronomy, Crop Science Society of America, Soil Science Society of America), IS-MPMI (International Congress on Molecular Plant-Microbe Interactions) and Plant breeding symposiums. We also collaborate with cotton field extension specialists. What do you plan to do during the next reporting period to accomplish the goals?Our research will continue as proposed advancing towards our goals in the original proposal. We are working towards the timely publication of our current research findings to the scientific community. Our next steps are completing analysis forGWAS study across 3 years of field screening data for different races and validating resistance gene candidates and segregating haplotypes, training Postdocs, graduate, and undergraduate students.

    Impacts
    What was accomplished under these goals? Reveal the Fov field genotypes at the nucleotide level. Draft contig-level genome assemblies of Race1 (CA10), Race4 (CA14), TX_17-3 (Race4), and more virulent Texas Race4 isolates (TX_18-24) were further analyzed using chromosome conformation capture with HiC sequencing in-order to obtain a complete chromosome-scale assembly. Complete chromosome assembly of these four genomes was highly contiguous with N50 ranging from 4.4 to 4.9Mb. Chromosome-scale assemblies of these four Fov genomes were also reannotated using MAKER annotation pipeline and RNASeq to obtain gene models. The resulting assemblies were aligned to the Fusarium oxysporum f. sp lycopersici (Fol) genome a well-annotated and researched tomato wilt pathogen. This revealed for the first time the unique additional chromosomes of Race1 and Race4 Fov isolates in addition to the eleven homologous chromosomes also known as the core chromosomes are conserved within the Fusarium Oxysporum Species Complex (FOSC). Race1 possessed 13 chromosomes, 2 of which are unique to Race1 with a total approximate genome size of 49.8Mb while Race4 possesses 17 chromosomes 5 of which are unique to Race4 with an approximate genome size of 61.5. Further splitting of core chromosome 12 is only observed in Race4 and designated as 12a and 12b. These unique additional small chromosomes in Race1 were designated as accessory chromosomes. Race4 has not only acquired more accessory chromosomes but has also increased significantly in size by about 10+ Mb compared to Race1. Further in-depth analysis into the core versus the new genomic regions for orphan genes was performed using blastp. The genome-wide orphan gene distribution was done for all 4 genomes with complete chromosome assembly. The accessory chromosomes possessed significantly more Orphan genes compared with the core chromosomes in all four. Orphan genes were further classified into putative effectors and are currently being optimized to obtain and design better classification tools for genome-wide field isolate classification and distinguishing between more virulent and less virulent Race4 field isolates. Accessory chromosomes are also more highly enriched in transposons and carry a fewer number of genes compared to the core chromosomes. Several gene duplication events are also observed in these accessory chromosomes. Thus, accessory chromosomes, genome expansion, gene duplication, orphan genes, and transposons could play an important role in pathogen adaptation and evolution. Evolutionary analysis for Fov races was performed and this revealed that Race1 isolate evolved independently of Race4. Resequencing of 31 additional Texas field Isolates grouped 92% of Texas field isolates into the Race4 clade regardless of virulence. Non-pathogenic Texas isolate TX_17-6 are classified as Fusarium graminaerum and TX_18-6 as Fusarium fujikoroi. Stacked SNP distribution of 27 Race4 Texas field isolates based on Reference Race 4 (CA14) shows that SNPs are more distributed on chromosomes 9, 10, 14, 15, and 18. Phylogenic placement of Fov within the FOSC was done using 82 whole-genome sequencing data and results show the relatedness of all the members in the complex. Our study also reveals the broad host range of Fov causing wilting, chlorosis, and stunting phenotypes in tobacco, okra, cilantro, cantaloupe, spring onion, Arabidopsis, and peas. ? Objective 2. Germplasm screens of upland cotton resistance/tolerance to Fov4 (Hague & Dever). Significant progress has been made towards germplasm screens of upland cotton lines 2.1: We increased the seed of over 200 multi-allelicadvanced generation intercross (MAGIC) Lines for to include in 2020 and 2021 field and lab screening. 2.2: We screened 125 lines over 5 separate tests in 2018 to evaluate the best modifications for field screening strategy. 2.3: All phenotypic evaluations were completed in 2018, with the final root staining value providing the best statistical separation among lines with varying resistance to Fov4. The best level and uniformity inoculum field location for 2019 were identified and additional 132 lines are in the field for both GWAS studies and cultivar development potential. 2.4: Fifty-six different RxR crosses were initiated in 2018 based on preliminary screening results, and F2 populations are growing in a Lubbock selection nursery and in Fov4-infested field evaluation currently in 2019. Additional evaluations are being made on RxS populations in Fov4-infested field in 2019. 2.5: Last year of GWAS lines phenotyping in Fov4-infested field was completed in 2020 and 2021. Phenotypic data is currently being analyzed for association studies Objective 3. Characterization of candidate Fov4 resistance genes introgressed into upland line and G. barbadense - Pima S-6 (He & Ulloa). Whole-genome sequencing results from 384 upland cotton lines have just been received and are currently being analyzed to detect significant loci and segregating haplotypes associated with resistance in upland cotton. Candidate genes and positive haplotypes resulting from the association studies will be further validated to ascertain their function and role in Fov resistance using the Virus-Induced Gene Silencing technique (VIGs) for cotton optimized in our lab.

    Publications

    • Type: Book Chapters Status: Published Year Published: 2021 Citation: Zunyong Liu, Eun-Gyu No, Catherine Danmaigona Clement, Ping He, Libo Shan Isolation of High-Molecular Weight (HMW) DNA from Fusarium oxysporum f. sp. vasinfectum for long-read sequencing. Fusarium wilt, Chapter 2, 1st ed. 2022 Methods and Protocols Methods in Molecular Biology Series, Vol. 2391
    • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Catherine Danmaigona Clement, Zunyong Liu, Lin Zhang, Steve Hague, Terry Wheeler, Jane K Dever, Ping He and Libo Shan. Deploying genomics in cotton disease outbreak. ASA, CSSA and SSSA International Annual Meetings Nov 2021 ***1st Place CO1 Crop Breeding and Genetics ($500) *** ***2nd Place Society-wide CSSA ($750) ***
    • Type: Websites Status: Published Year Published: 2021 Citation: Marc Zienkiewicz (2021 October). The daughter of a Great Farmer. Seed World, p. 40 https://seedworld.com/flipbook_october2021/?page=42
    • Type: Websites Status: Published Year Published: 2021 Citation: Marc Zienkiewicz (2021 August). Catherine Danmaigona Clements Love of Agriculture Runs in the Family. https://seedworld.com/catherine-danmaigona-clements-love-of-agriculture-runs-in-the-family/
    • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Catherine Danmaigona Clement, Zunyong Liu, Lin Zhang, Steve Hague, Terry Wheeler, Jane K Dever, Ping He, and Libo Shan. Genomic Plasticity and Virulence Diversity of Fusarium oxysporum f. sp. vasinfectum Race4 from Cotton Fields. ASA, CSSA, and SSSA International Annual Meetings Nov 2021
    • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Catherine Danmaigona Clement, Zunyong Liu, Lin Zhang, Steve Hague, Terry Wheeler, Jane K Dever, Ping He, and Libo Shan Comparative genomics and infection process of Fov (Fusarium oxysporum f. sp. vasinfectum) in cotton. University of Minnesota Plant breeding symposium 2021. ***2021 Student speaker finalist***
    • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Catherine Danmaigona Clement, Zunyong Liu, Lin Zhang, Steve Hague, Terry Wheeler, Jane K Dever, Ping He, and Libo Shan. Comparative genomics and evolutional history of Fov (Fusarium oxysporum f. sp. vasinfectum) in US cotton reveal Race specific regions related to pathogenicity. Plant breeding circle, Department of Soil and Crop Sciences Texas A&M University 2021.
    • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Catherine Danmaigona Clement, Zunyong Liu, Lin Zhang, Eun-Gyu No, Steve Hague, Terry Wheeler, Jane K Dever, Ping He and Libo Shan. Dissecting the Genome Structure of Fusarium oxysporum f. sp Vasinfectum Race4 Causing Devastating Losses in Cotton. ASA, CSSA and SSSA International Annual Meetings 2021. ***1st Place society-wide diversity poster contest ($300) *** ***1st Place CO1 Crop Breeding and Genetics ($500) ***
    • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Catherine Danmaigona Clement, Zunyong Liu, Lin Zhang, Eun-Gyu No, Steve Hague, Terry Wheeler, Jane K Dever, Ping He and Libo Shan. Genomic plasticity and virulence diversity of Fusarium oxysporum f. sp. vasinfectum (Fov) from cotton fields. International Society for Molecular Plant-Microbe Interactions conference (IS-MPMI) 2021.
    • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Catherine Danmaigona Clement, Lin Zhang, Zunyong Liu, Steve Hague, Terry Wheeler, Jane K Dever, Ping He and Libo Shan. Comparative Genomics of Fov (Fusarium oxysporum f. sp. vasinfectum) in US Cotton Provides Insights into Race-Specific Regions Related to Pathogenicity and Evolution. Cotton Beltwide conference 2021.


    Progress 07/15/19 to 07/14/20

    Outputs
    Target Audience:Cotton scientific community, the general scientific community, and agricultural-related sectors" Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has made available several avenues for training postdoctoral fellows, graduate students, and undergraduates. How have the results been disseminated to communities of interest?Findings are disseminated primarily through peer-reviewed journal articles, presentations, conferences and symposium. We also participated in the cotton breeders tour and collaborated with cotton field extension specialists. What do you plan to do during the next reporting period to accomplish the goals?Our research will continue as proposed advancing towards our goals in the original proposal. We are working towards timely publication of our research findings to the scientific community, training Postdocs, graduate, and undergraduate students.

    Impacts
    What was accomplished under these goals? Reveal the Fov field genotypes at the nucleotide level. We accomplished this goal during this year. Texas field in El Paso was surveyed and Fov was isolated from diseased cotton plants from the year of the outbreak for two years. Thirty-two (32) Isolates from Infected cotton fields in 2017 was provided by Dr. Jason Woodward and twenty-two (22) Isolates from El Paso for year 2018 were provided by Dr. Tom Isakeit. For initial classification, fragments of four nuclear genes, translation elongation factor (EF-1α), Beta tubulin (BT), phosphate permease (PHO), and Intergenic spacer (IGS) region were used to sequence all the isolates. Primers were designed to span the conserved regions for isolates distinctions. Initial grouping of isolates was visualized using phylogenic trees. Our results show some isolates were like CA10 (Race 1), Race3 and CA14 (Race 4) but several isolates were unclassified based on the available marker genes classification used by the cotton community. From the results of this initial marker classification, isolates were selected from different clades and whole genome sequencing of 30 Fusarium isolates were re-sequenced by Illumina at 98-148X for accurate classification at the whole genome level and population genetics study. Our result based on whole genome sequencing data accurately classified the Fov isolates in our study and reveals some misclassification from the initial grouping using the four marker genes. From an evolutionary standpoint our results reveal that, CA10 (Race 1) is closest to the Ancestral genome, followed by Fov Race3, Fov Race8 and then CA14 (Race4). All the Race4 Isolates regardless of how virulent they are were classified as Race 4 because they were all in the same clade as CA14 except for TX_17-6 and TX_18-6. Our study also established that some Texas Isolates were more virulent than the isolates from California. In order to understand and capture the virulence of different isolates we looked at the whole genome SNP distribution with TX_17-3 isolate as reference. We had established that TX_18-24 and TX_18-9 isolates are more virulent than CA14 and TX_17-6 and TX_17-24 isolates were not virulent against cotton genotypes. Phylogeny from whole genome SNP distribution grouped the isolates based on virulence and new markers can be designed from these SNPs for more accurate classification of Fov isolates. Lineage-specific chromosomes have been reported to determine host specific pathogenicity in Fusarium oxysporum species but there is no report of lineage specific chromosomes in Fov and its function. We deployed long read sequencing and performed a denovo genome assembly, annotation and detected race-specific regions unique to Race1 and Race4 Fov genomes. Upon further analysis, these Race-specific regions were more enriched in GC content, repetitive sequences, transposons and effectors compared to the core regions of all four genomes sequenced. It is well known that fungal avirulence genes encode virulence factors called effectors which play major role in virulence. Race-specific chromosomes amassed more secreted effectors with transmembrane domain and extracellular localization in contrast with the core chromosome regions of Fov. Genes and transposon elements enriched in these Race-specific regions might have strong correlation with pathogenicity and might explain the different behavior of CA10 (Race 1) Fov isolates compared to CA14 (Race 4) isolates. We also detected several gene duplication only observed in the Race-specific regions of the Fov genomes which plays an important role in the evolution. This is the first report of compartmentalization and identification of Race-specific regions with several events of gene duplication in the Fov genome. Investigate Fov colonization process in cotton roots of resistant and susceptible lines. This objective was completed during this year. We deployed susceptible cotton line PHY 499WRF and tolerant cotton line PHY 881FR to observe the infection process and colonization of cotton root tissues upon infection with Fov. PHY499WFR were also inoculated and grown in hydroponics and time course of infection was observed from day of inoculation to wilting and eventual death of cotton plant. We observed Fov spores germination and hyphae penetrating in the plant cells from intercellular space at 10 hours, and the hyphae were limited within the plant cells (12hpi). Strands of hyphae continues to grow and expand within the plant intercellular spaces of the cotton plant (48hpi, 72hpi, 5dpi). Network of hyphae is detected growing in the central cylinder of the root and colonizing the intercellular space of cortex cells (7dpi) and vascular tissues (14dpi). At 20dpi multiple strands of hyphae and germinated conidia is apparent within the xylem vessels with overwintering structures formed in dead plants. Time course of infection was also observed in a Tolerant Pima cotton PHY881FR using a similar method for PHY499. Phenotype of infected seeds shows no visual sign of Fov infection. Seeds were not inhibited from germination and develop fully expanded leaves. The surface of germinated radicle shows slight signal of elongating of Fov parallel to the longitudinal axis of the radicle. Surface of germinated seedlings shows slight growth of fungal hyphae at 48hpi for both Fov Isolates. At 96hpi, fluorescing Fov hyphae multiplies and is detected on the surface of germinated pima cotton but with no hyphae signal in apparent within central cylinder or the xylem tissues of cotton seedling for both Fov Isolates. At the 7days time point, fluorescing continued colonization of the surface of germinated seedlings and root surfaces was detected for both Fov isolates. Hyphae strands or growth was not detected in the cortex or vascular tissues of the seedling. Objective 2. Germplasm screens of upland cotton resistance/tolerance to Fov4 (Hague & Dever). Significant progress has been made towards germplasm screens of upland cotton lines 2.1: We increased seed of over 500 lines based on preliminary screening in 2017 to include in 2018 and 2019 field screening. 2.2: We screened 125 lines over 5 separate tests in 2018 to evaluate best modifications for field screening strategy. 2.3: All phenotypic evaluations were completed in 2018, with final root staining value providing the best statistical separation among lines with varying resistance to Fov4. Best level and uniformity inoculum field location for 2019 was identified and additional 132 lines are in the field for both GWAS studies and cultivar development potential. 2.4: Fifty-six different RxR crosses were initiated in 2018 based on preliminary screening results, and F2 populations are growing in a Lubbock selection nursery and in Fov4-infested field evaluation currently in 2019. Additional evaluations are being made on RxS populations in Fov4-infested field in 2019. 2.5: Second year of GWAS lines phenotyping in the Fov4-infested field currently in 2019. Cotton lines in the GWAS panel are currently being screened for Race1 resistance in the greenhouse and tissue for high molecular weight DNA extraction have also been collected for Illumina resequencing. We also report for the first time a fast and efficient protocol for rapid screening of Cotton lines to Fov resistance eliminating 90% of susceptible line within 2 days saving time and resources required for large-scale field screening. This is an efficient method to use as a first step in detecting susceptible lines before further greenhouse or field screening. Results from this method have been consistent. Infection process of the seeds using this method was verified using GFP tagged Fov strains for validation.

    Publications