Research Project: Disease Management and Improved Detection Systems for Control of Pathogens of Vegetables and Strawberries

Location: Crop Improvement and Protection Research

2023 Annual Report

Objectives
This project will create new ways to manage plant diseases of strawberries and vegetables. New approaches to disease management and resistance breeding are needed to protect crops from diseases, including increased knowledge of pathogen detection, disease cycles, and pathogen and plant genomes, as well as the potential for soil microbes that can be utilized for disease suppression. This project will lead to disease control with reduced fungicides and fumigants and will use both agricultural automation and traditional approaches. We will focus on the following major objectives and subobjectives during the next five years. Objective 1: Integrate understanding of pathogen genomes into new tools for pathogen diagnostics. Sub-objective 1.A: Develop molecular markers for diagnostic assay development for formae speciales of Fusarium oxysporum by analysis of genomic sequence data. Sub-objective 1.B: Develop diagnostic markers and tools for rapid identification of oomycetes using comparative analysis of mitochondrial genomes. Sub-objective 1.C: Determine if strawberry-pathogenic isolates of Macrophomina phaseolina are polyphyletic within the context of the broad diversity of this species. Objective 2: Gain a better understanding of strawberry and vegetable pathogen life cycles and how microbial communities can be used for new management strategies. Sub-objective 2.A: Identify genes required for microsclerotia formation in Verticillium dahliae. Sub-objective 2.B: Functionally characterize the role of a predicted pathogenicity chromosome in F. oxysporum f. sp. fragariae on the strawberry host-specific pathogenicity phenotype. Sub-objective 2.C: Investigate the role of oospores in the life cycle of Peronospora effusa. Sub-objective 2.D: Determine if genetically diverse Macrophomina phaseolina isolates from strawberry exhibit differential virulence on tolerant strawberry varieties. Sub-objective 2.E: Identify the role of the strawberry root microbiome in Verticillium wilt of strawberry. Objective 3: Integrate management strategies for improved control of strawberry and vegetable pathogens. Sub-objective 3.A: Determine if an avirulence gene identified in race 1 strains of F.o. fragariae is the avirulence gene specific for FW1-mediated resistance (called AvrFW1). Sub-objective 3.B: Characterize gene expression in spinach-downy mildew interactions in response to two races of Peronospora effusa. Sub-objective 3.C.1: Identify the effect of biofumigant green manure soil amendment type and application rate on soil microbiome function and soilborne pathogen inoculum survival. Sub-objective 3.C.2: Identify the effect of pre-plant application of antimicrobial producing Streptomyces bacteria on Fusarium wilt diseases of vegetables. Sub-objective 3.D.1: Evaluate nitrogen dioxide (NO2) as a seed treatment for two pathogens carried on spinach seeds. Sub-objective 3.D.2: Investigate applications of automation technology for use in strawberry production systems toward improving production practices and control of pests and diseases. Sub-objective 3.D.3: Identify alternative soil fumigants to control soilborne pathogens and nematodes in carrot and other crops.

Approach
Sub-objective 1.A: Unique sequences suitable as targets for developing taxon specific markers will be identified in genomic sequence data. Sub-objective 1.B: Mitochondrial genomes will be used for selection of loci for development of diagnostic markers. Sub-objective 1.C: Phylogenies will reveal if strawberry-pathogenic strains of M. phaseolina are polyphyletic within the broader diversity of this species. Sub-objective 2.A: Comparative analyses of genomes of Verticillium species will reveal sequences required for microsclerotia formation. Sub-objective 2.B: The loss of the chromosome will be induced and/or transferred to a non-pathogenic strain, and disease examined. Sub-objective 2.C: Oospores of P. effusa will be examined for their ability to infect roots or leaves in growth chambers and humidity tents. Sub-objective 2.D: A broad diversity of isolates will be tested for pathogenicity on 4 tolerant and 2 susceptible varieties. Sub-objective 2.E: We will assess if microbial taxa in the root microbiome that correlates with pathogen biomass in the roots or disease severity significantly affects Verticillium wilt of strawberry. Sub-objective 3.A: Quantitative trait loci (QTL) and genome-wide association studies(GWAS) to map genomic regions associated with resistance. Sub-objective 3.B: RNASeq analysis of gene expression will be performed. Sub-objective 3.C.1: Five weed or crop species will be tested against Verticillium dahliae as biofumigants. Sub-objective 3.C.2: Biological control will be tested by applying Streptomyces spores to field soil as pre-plant treatments for control of Fusarium wilt. Sub-objective 3.D.1: Varying percent of NO2 will be applied in chambers with seeds harboring two pathogens. Sub-objective 3.D.2: Approaches using automation technology will be assessed for use in strawberry production systems to improve disease control. Sub-objective 3.D.3: Assess anaerobic soil disinfestation and incorporation of green manures to control soilborne pathogens and nematodes in California carrot and other cropping systems.

Progress Report
In support of Sub-objective 1.A, we generated Illumina genomic sequence data from over 370 isolates of Fusarium oxysporum to add to our sequence database, which currently represents approximately 59 formae specialis and multiple races of specific taxa. Three different software pipelines using different approaches were developed for identification of unique regions to target for development of molecular diagnostic assays. This sequence data and software pipelines have been used to develop and field validate a molecular diagnostic marker for F. oxysporum f. sp. lactucae Race 1, which is a major emerging pathogen of lettuce. Assays were developed using both TaqMan real time polymerase chain reaction (PCR) and the isothermal amplification technology called Recombinant Polymerase Amplification (RPA). The technique has been shared with research and commercial labs, and a manuscript will soon be ready for submission. We extracted 41 phylogenetically informative genes from all isolates in the database and provided them to a collaborator at Pennsylvania State University for conducting a comprehensive multilocus analysis of F. oxysporum. The manuscript is in the final stages of preparation. Using the 41 gene dataset from the phylogenetic analysis a new identification technique was developed that uses sequence data from five different loci to classify isolates. This technique presents a significant improvement for delineating isolates over the current approach that relies primarily on one gene. The manuscript is ready for submission pending completion of the phylogenetic manuscript. In support of Sub-objective 1.B, genomic Illumina data has been generated from additional oomycete species and mitochondrial genomes assembled. In collaboration with a lab at the University of California (UC), Davis, the primary focus has been tropical graminicolous downy mildews primarily in the genus Peronosclerospora. Between historical collections, herbarium samples and contemporary collections by collaborators, data has been generated for over 272 samples with mitochondrial genomes at some point of assembly for the vast majority. The gene data is being used to investigate the phylogeny of the genus as well as clarify species boundaries, in particular for the select agent P. philippinensis. Diagnostic markers for Peronosclerospora at a genus level were developed with species specific markers currently being validated for several taxa, including P. philippinensis. In addition, simple sequence repeats (SSR) markers and mitochondrial haplotype markers were developed for conducting population studies of this species. Our collaborator at UC Davis is focusing on assembly and annotation of the nuclear genomes. In support of Sub-objective 1.C, we collected more than 400 isolates of Macrophomina spp. from 27 countries. Most of these isolates were M. phaseolina, but a few represented newly described species M. tecta, M. pseudophaseolina, and M. euphorbiicola. All of these isolates were whole genome shotgun sequenced and variants were called and analysis of their content has begun. The initial results indicate that M. phaseolina isolates derived from diseased strawberry plants are polyphyletic, although most are in a single clade. In support of Sub-objective 2.A, comparative genomics analyses were performed using computer programs and genomic sequences of different Verticillium species that produce or do not produce microsclerotia. From these results, a list of candidate genes were identified for experimental analyses to assess which genes may be important for microsclerotia production in some Verticillium species. As part of the initial work to accomplish Sub-objective 2.B, we developed additional isolates that are resistant to the antibiotic hygromycin and have the hygromycin resistance gene incorporated into a putative pathogenicity chromosome. This will allow us to determine if the benomyl treatment is inducing loss of the putative pathogenicity chromosome (by testing for susceptibility to hygromycin). As part of the initial work to accomplish Sub-objective 2.C, oospores of the spinach pathogen Peronospora effusa were collected from leaf tissue, and spinach seedlings were inoculated and evaluated for disease development. Additionally, we determined that oospores obtained from spinach leaves gave rise to downy mildew on spinach when mixed with seeds at planting. The replicated experiments were performed in glass chambers in which soil was fumigated to eliminate the possibility of air or soil contamination prior to the experiments. Additional experiments also confirmed seed transmission of downy mildew disease from oospore-infested seeds. In support of Sub-objective 2.D, we identified genetically distinct isolates of Macrophomina phaseolina derived from diseased strawberry plants and tested them for virulence on strawberry. We are first testing them for virulence on susceptible strawberry varieties before moving to testing them on broader arrays of cultivars. In support of Sub-objective 2.E, we generated Verticillium dahliae microsclerotia through infection and harvesting of a susceptible lettuce cultivar. We also established strawberry nursery transplants inoculated with V. dahliae in field soil. As part of initial work to accomplish Sub-objective 2.E, V. dahliae inoculum was generated and strawberry nursery transplants were inoculated with V. dahliae and transplanted into field soil. A second replicate experiment was also established using the same methods to evaluate reproducibility of the effects of V. dahliae on strawberry root microbiomes. In support of Sub-objective 3.A, we generated seedlings from a cross between a resistant and a susceptible strawberry variety. The resistant variety was heterozygous for the dominant allele of the FW1 resistance locus while the susceptible variety was homozygous for the susceptible allele. More than 200 progeny were tested for susceptibility to the AvrFW1 knockout strain of Fusarium oxysporum f. sp. fragariae; this gene knockout confers virulence on cultivars with FW1. Each progeny was also genotyped for FW1 and we are currently analyzing the results. In support of Sub-objective 3.B, we collected leaf tissue from downy mildew resistant and susceptible spinach plants inoculated with two different races of Peronospora effusa and performed RNA-Sequencing and initial data analyses. As part of the initial work to accomplish Sub-objective 3.D, new seed samples were obtained from commercial spinach seed lots and examined and found to have oospores for the spinach downy mildew pathogen Peronospora effusa and microsclerotia of the Verticillium wilt pathogen, Verticillium dahliae. Nitrogen dioxide gas was used to assess the elimination of V. dahliae in intact spinach seeds and those that were ground. In support of Sub-objective 3.C.1, we generated Verticillium dahliae microsclerotia through infection and harvesting of a susceptible lettuce cultivar. As part of initial work to accomplishment Sub-objective 3.C.1, V. dahliae microsclerotia were generated and used to infest soil. In support of Sub-objective 3.C.2, we sampled soil from multiple locations and isolated Streptomyces bacteria. As part of initial work to accomplish Sub-objective 3.C.2, soil was sampled from agricultural fields in Monterey and Santa Cruz Counties. Putative Streptomyces bacteria were isolated from soil samples.

Accomplishments
1. Soilborne oospores of the spinach downy mildew pathogen can initiate disease. Spinach downy mildew, caused by Peronospora effusa, is a major disease on spinach in the United States and worldwide. ARS researchers in Salinas, California, and Fort Detrick, Maryland, determined that oospores obtained from spinach leaves gave rise to downy mildew on spinach when mixed with seeds at planting. The replicated experiments were performed in glass chambers in which soil was fumigated to eliminate the possibility of air or soil contamination prior to the experiments. Additional experiments also confirmed seed transmission of downy mildew disease from oospore-infested seeds. These results are used by spinach growers to address the spinach disease threat posed by soilborne and seedborne oospores.

2. Uncovering regulatory mechanisms for melanin and microsclerotia production in Verticillium dahliae. Verticillium dahliae is a plant pathogenic fungus that penetrates plant roots and invades the water conducting xylem tissue of plants to cause disease symptoms of chlorosis and wilt. This fungus survives long term in soils by producing survival structures known as microsclerotia, which are heavily melanized. ARS researchers in Salinas, California, identified a gene in V. dahliae encoding a protein that that acts as a powerful negative regulator of microsclerotia and melanin production, and mutation of this gene also rendered the fungus defective in pathogenicity. Identification of the genes that control aspects of survival, asexual reproduction, and virulence in V. dahliae led to alternative disease control measures.

3. Beneficial microorganisms increase yield in commercial strawberry production systems. Beneficial microorganisms can improve strawberry plant health and reduce the need to use soil fumigants. ARS researchers in Salinas, California, collaborated with a strawberry grower to test the effect of commercially available beneficial microorganisms on strawberry under field conditions. Strawberry plants inoculated with beneficial microorganisms produced significantly larger strawberries compared to non-treated plants. Results from this research are being used by growers who are interested in using beneficial microorganisms to increase strawberry yield in conventional and organic production systems.

4. Bacteria from healthy strawberry suppress strawberry plant growth. Strawberry plants are naturally colonized by many different microorganisms. Identifying how these microorganisms impact plant growth is needed to predict and improve crop yield. ARS researchers in Salinas, California, isolated and identified a novel bacterial species from roots of healthy strawberry plants. Re-infection of strawberry plants with the bacterium reduced plant growth, but did not lead to any overt symptoms of disease. These results suggest microorganisms that naturally colonize strawberry plants and do not cause disease can still reduce crop yield and may be an overlooked problem in strawberry production systems. This information will be useful to growers looking to increase strawberry yield, even in the absence of clear disease pressure.

5. Discovered a resistance-breaking strain of the Fusarium wilt of strawberry pathogen. Fusarium wilt of strawberry, caused by Fusarium oxysporum f. sp. fragariae, is an economically damaging disease that is most effectively and economically controlled by growing a disease-resistant cultivar. Multiple cultivars have resistance to this pathogen conferred by a dominant gene at a single locus, called FW1. Previously, FW1 was considered effective against all strains of this pathogen in California. That changed this year when an ARS scientist at Salinas, California, discovered for the first time a resistance breaking strain of Fusarium oxysporum f. sp. fragariae in a California fruit production field. The early detection of this new strain has enabled the strawberry industry to launch new efforts to breed for resistance, enact a statewide surveillance program, and attempt to contain the pathogen at the site of its emergence.

6. Identified an airborne spore dispersal phase of F. oxysporum f.sp. fragariae. The pathogen Fusarium oxysporum f.sp. fragariae has always been considered soilborne and only trafficked between fields with soil or infected plant material. This year an ARS scientist at Salinas, California, discovered growth of Fusarium oxysporum f. sp. fragariae on dead tissue above-ground. This type of growth has rarely been observed in plants infected by Fusarium wilt pathogens, but where it occurs it is always associated with the production of airborne spores. The ARS scientist in Salinas, California, confirmed this tissue could produce airborne spores and that it was common on infected strawberries in California. Strawberry growers are reducing crop loss to this disease by avoiding field preparation practices that are vulnerable to infestation by airborne spores.

7. Determined the host range of Macrophomina phaseolina has been consistently exaggerated in the scientific literature. Few aspects of plant pathogen biology are as important as host range, or the specific crops that are susceptible to disease. It is commonly stated in the literature that Macrophomina phaseolina has a host range of over 500 plant species, but ARS scientists in Salinas, California, noticed that no citation could provide support for this claim. Through a comprehensive literature meta-analysis of more than 900 peer-reviewed publications, ARS scientists discovered the claim was initially based on an obsolete definition of the term ‘host range’ and that the true, documented host range of M. phaseolina includes only about 100 plant hosts. Agricultural producers now avoid crop rotations with susceptible hosts and scientists are focusing on overlooked aspects of this pathogen’s biology.

8. Improved DNA extraction from soil for enhanced disease risk assessment. The ability to quantify soilborne fungal plant pathogens in a field prior to planting would help growers assess risk and make management decisions to reduce losses. However, for many fungal taxa techniques to facilitate this are not available. An ARS scientist in Salinas, California, has developed molecular assays capable of quantifying several of the important pathogens of strawberry and vegetable crops often grown in rotation as well as an improved soil DNA extraction procedure that results in purified DNA from larger amounts of soil than previously used. These accomplishments, and work to correlate the results of the molecular assays to inoculum levels in the soil, have improved our ability to accurately quantify lower levels of the pathogens and thereby provide more accurate risk assessment data to growers.

Review Publications
Xiao, L., Tang, C., Klosterman, S.J., Wang, Y. 2023. VdTps2 modulates plant colonization and symptom development in Verticillium dahliae. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-03-23-0024-R.
Zima, H.V., Clark, K.J., Poudel-Ward, B., Slinksi, S.L., Klosterman, S.J., Correll, J.C. 2022. Evaluation of spinach cultivars for downy mildew resistance in Yuma, AZ 2022. Plant Disease Management Reports. 16. https://doi.org/10.1094/PDMR16.
Zhang, D., Dai, X., Klosterman, S.J., Subbarao, K.V., Chen, J. 2022. The secretome of Verticillium dahliae in collusion with plant defence responses modulates Verticillium wilt symptoms. Biological Reviews. 97(5):1810-1822. https://doi.org/10.1111/brv.12863.
Li, H., Wang, D., Zhang, D.D., Geng, Q., Li, J.J., Sheng, R.C., Xue, H.S., Zhu, H., Kong, Z.Q., Dai, X.F., Klosterman, S.J., Subbarao, K.V., Chen, F.M., Chen, J.Y. 2022. A polyketide synthase from Verticillium dahliae modulates melanin biosynthesis and hyphal growth to promote virulence. BMC Biology. 20. Article 125. https://doi.org/10.1186/s12915-022-01330-2.
Wang, T., Shaban, M., Shi, J., Wang, W., Liu, S., Nie, X., Yu, Y., Kong, J., Klosterman, S.J., Zhang, X., Aierxi, A., Zhu, L. 2022. Attenuation of ethylene signaling increases cotton resistance to a defoliating strain of Verticillium dahliae. The Crop Journal. 11(1):89-98. https://doi.org/10.1016/j.cj.2022.05.008.
Tian, L., Zhuang, J., Li, J.J., Zhu, H., Klosterman, S.J., Dai, X.F., Chen, J.Y., Subbarao, K.V., Zhang, D.D. 2023. Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae. Frontiers in Microbiology. 14. Article 1130468. https://doi.org/10.3389/fmicb.2023.1130468.
Li, R., Xue, H.S., Zhang, D.D., Wang, D., Song, J., Subbarao, K.V., Klosterman, S.J., Chen, J.Y., Dai, X.F. 2022. Identification of long non-coding RNAs in Verticillium dahliae following inoculation of cotton. Microbiology Research. 257. Article 126962. https://doi.org/10.1016/j.micres.2022.126962.
Miao, Y., Chen, K., Deng, J., Zhang, L., Wang, W., Kong, J., Klosterman, S.J., Zhang, X., Aierxi, A., Zhu, L. 2022. miR398b negatively regulates cotton immune responses to Verticillium dahliae via multiple targets. The Crop Journal. 10(4):1026–1036. https://doi.org/10.1016/j.cj.2021.12.010.
Li, C., Qin, J., Huang, Y., Shang, W., Chen, J., Klosterman, S.J., Subbarao, K.V., Hu, X. 2023. The Verticillium dahliae secreted protein VdCE11 contributes to virulence by promoting accumulation and activity of the cotton aspartic protease GhAP1. Microbiology Spectrum. 11(1). https://doi.org/10.1128/spectrum.03547-22.
Xiao, S., Ming, Y., Hu, Q., Ye, Z., Si, H., Liu, S., Zhang, X., Wang, W., Yu, Y., Kong, J., Klosterman, S.J., Lindsey, K., Zhang, X., Aierxi, A., Zhu, L. 2023. GhWRKY41 forms a positive feedback regulation loop and increases cotton defense response against Verticillium dahliae by regulating phenylpropanoid metabolism. Plant Biotechnology Journal. 21(5):961-978. https://doi.org/10.1111/pbi.14008.
Rogers, L.W., Koehler, A.M., Crouch, J.A., Cubeta, M.A., LeBlanc, N.R. 2022. Comparative genomic analysis reveals contraction of gene families with putative roles in pathogenesis in the fungal boxwood pathogens Calonectria henricotiae and C. pseudonaviculata. BMC Ecology and Evolution. 22:29. https://doi.org/10.1186/s12862-022-02035-4.
LeBlanc, N.R., Gebben, S. 2023. Soil bacterial communities are influenced by soil chemical characteristics and dispersal limitation in commercial strawberry production systems. Plant-Environment Interactions. 4(1):11-22. https://doi.org/10.1002/pei3.10099.
Henry, P.M., Koehler, S., Kaur, S., Epstein, L., Mitchell, J.P., Gordon, T.R., Leveau, J.H. 2022. Amplicon sequencing of Fusarium translation elongation factor 1a reveals that soil communities of Fusarium species are resilient to disturbances caused by crop and tillage practices. Phytobiomes Journal. 6(3):261-274. https://doi.org/10.1094/PBIOMES-09-21-0053-R.
Epstein, L.E., Kaur, S., Henry, P.M. 2022. The emergence of Fusarium oxysporum f. sp. apii race 4 and Fusarium oxysporum f. sp. coriandrii highlight major obstacles facing agricultural production in coastal California in a warming climate: A case study. Frontiers in Plant Science. 13. Article 921516. https://doi.org/10.3389/fpls.2022.921516.
Steele, M.E., Hewavitharana, S.S., Henry, P.M., Goldman, P.H., Holmes, G.J. 2023. Survey of late-season soilborne pathogens infecting strawberry in Watsonville-Salinas, California. Plant Health Progress. 24(1):104-109. https://doi.org/10.1094/PHP-06-22-0056-S.
Jenner, B.N., Henry, P.M. 2022. Pathotypes of Fusarium oxysporum f. sp. fragariae express discrete repertoires of accessory genes and induce distinct host transcriptional responses during root infection. Environmental Microbiology. 24(10):4570-4586. http://doi.org/10.1111/1462-2920.16101.
Cespedes, M.K., Melgarejo, T.A., Henry, P.M., Rwahnih, M.A., Gilbertson, R.L. 2022. First report of watermelon mosaic virus naturally infecting coriander (Coriandrum sativum) and causing a leaf mottling disease in California. Plant Disease. 107(4):1248. https://doi.org/10.1094/PDIS-05-22-1184-PDN.
Espindola, A.S., Cardwell, K., Martin, F.N., Hoyt, P.R., Marek, S.M., Schneider, W., Garzon, C.D. 2022. A step towards validation of high-throughput sequencing for the identification of plant pathogenic oomycetes. Phytopathology. 112(9):1859-1866. https://doi.org/10.1094/PHYTO-11-21-0454-R.
Bello, J.C., Higgins, D.S., Sakalidis, M.L., Quesada-Ocampo, L.M., Martin, F.N., Hausbeck, M.K. 2022. Clade-specific monitoring of airborne Pseudoperonospora spp. sporangia using mitochondrial DNA markers for disease management of cucurbit downy mildew. Phytopathology. 112(10):2110-2125. https://doi.org/10.1094/PHYTO-12-21-0500-R.
Nguyen, H.D., Dodge, A., Dadej, K., Rintoul, T.L., Ponomareva, E., Martin, F.N., de Cock, A.W., Levesque, C.A., Redhead, S.A., Spies, C.F. 2022. Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium. Mycologia. 114(3):501-515. https://doi.org/10.1080/00275514.2022.2045116.
Botkin, J.R., Chanda, A.K., Martin, F.N., Hirsch, C.D. 2022. A reference genome sequence resource for the sugar beet root rot pathogen Aphanomyces cochlioides. Molecular Plant-Microbe Interactions. 35(8):706-710. https://doi.org/10.1094/MPMI-11-21-0277-A.
Crouch, J.A., Davis, W.J., Shishkoff, N., Castroagudin, V.L., Martin, F.N., Michelmore, R., Thines, M. 2022. Peronosporaceae species causing downy mildew diseases of Poaceae, including nomenclature revisions and diagnostic resources. Fungal Systematics and Evolution. 9(1):43-86. https://doi.org/10.3114/fuse.2022.09.05.
Foster, Z.S., Albornoz, F.E., Fieland, V.J., Larsen, M.M., Jones, F.A., Tyler, B.M., Nguyen, H.D., Burgess, T., Riddell, C., Voglmayr, H., Martin, F.N., Grunwald, N.J. 2022. A new oomycete metabarcoding method using the rps10 gene. Phytobiomes Journal. 6(3):214-226. https://doi.org/10.1094/PBIOMES-02-22-0009-R.
Geiser, D., Martin, F.N., Espindola, A.S., Brown, J.K., Bell, T., Yang, Y., Kang, S. 2023. Knowledge gaps, research needs, and opportunities in plant disease diagnostic assay development and validation. PhytoFrontiers. 3(1):51-63. https://doi.org/10.1094/PHYTOFR-05-22-0057-FI.
Groth-Helms, D., Rivera, Y., Martin, F.N., Arif, M., Sharma, P., Castlebury, L.A. 2023. Terminology and guidelines for diagnostic assay development and validation: Best practices for molecular tests. PhytoFrontiers. 3(1):23-35. https://doi.org/10.1094/PHYTOFR-05-22-0059-FI.