The Diversity of Passalora fulva Isolates Collected from Tomato Plants in U.S. High Tunnels

Tomatoes (Solanum lycopersicum L.) are a high value crop in the United States with a farm gate value of over US$418 million. Between 2007 and 2019, the number of farms growing tomatoes in protected environments nearly tripled in the United States, totaling almost 7,974 farms in 2017 (USDA-NASS 2009, 2019). With the increasing use of high tunnels and greenhouses for season extension in the Northeast and Midwest, tomato leaf mold (caused by the biotrophic fungus Passalora fulva syn. Cladosporium fulvum) is now seen every year. The disease has become a management concern and can result in loss of yield, defoliation, and plant death (Thomma et al. 2005).

Detailed research from the 1930s (Bond 1938) describes infection by P. fulva occurring after conidia germinate on the abaxial leaf surface. The hyphae enter and block the stomata, which prevents the plant from respiring, eventually resulting in defoliation. Disease symptoms first appear as light-green spots on leaves. Subsequently, olive-green conidia emerge and eventually, the leaves show signs of necrosis (Fig. 1). Conidia disperse by water-splash or wind. It is most challenging to manage the disease in greenhouses or high tunnels, where conditions are humid (Thomma et al. 2005). The existence of a sexual stage has been hypothesized, but only the asexual stage has been observed (Stergiopoulos et al. 2007a, b)

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The increased use of high tunnels for tomato production in the United States, combined with the ability of P. fulva to rapidly adapt to and overcome resistance, has resulted in a problematic reoccurrence of tomato leaf mold. Beginning in the late 1800s, greenhouse production of tomatoes increased worldwide. Breeding for resistance became a priority, as fungicides were costly and not as effective as using resistant cultivars (Alexander 1934). By the 1930s, tomato breeders in the United States and Canada introgressed resistance genes (known as Cf genes for C. fulvum) from resistant wild tomato species, after it was observed that the red currant tomato (S. pimpinellilfolium) exhibited resistance to isolates of P. fulva (Alexander 1934; Langford 1937; Rivas and Thomas 2005). For several decades, outbreaks of leaf mold encouraged continued breeding efforts (Bailey 1950; Kerr and Bailey 1966; Rivas and Thomas 2005), as resistance was often overcome because of the strong selection after the introduction of the tomato Cf genes (Iida et al. 2015; Stergiopoulos et al. 2007b). In the United States and Canada, leaf mold resistance breeding activity declined after the 1970s, as attention shifted away from greenhouse production of tomatoes. Only in the last few decades, as greenhouse and high tunnel production has increased once again, have concerns over management of tomato leaf mold resurged.

The well-characterized effector proteins Avr2, Avr4, Avr4E, Avr5, and Avr9 are secreted by P. fulva during infection, and are recognized by the products of the single dominant resistance genes Cf-2, Cf-4, Cf-4E, Cf-5, and CF-9, respectively (Mesarich et al. 2014; Stergiopoulos and De Wit 2009). Although the study of pathogen race was common and began in the 1930s (Langford 1937), work has aimed to characterize pathogen populations once again, using differential sets of tomatoes for the determination of isolate race (Iida et al. 2010, 2015; Li et al. 2015; Lucentini et al. 2021; Medina et al. 2015; Rollan et al. 2013; Yoshida et al. 2021). In Japan, 13 races of P. fulva have been identified (Iida et al. 2015; Yoshida et al. 2021). Substantially fewer races of P. fulva were identified in Argentina, with all isolates belonging to either race 0 or 2 (Medina et al. 2015; Rollan et al. 2013). Earlier studies included tomatoes with Cf-1 and Cf-3 as part of the differential sets for race determination; however, studies do not include either resistance gene, as neither follow typical gene-for-gene interactions with effector complements (Boukema and Garretsen 1975; Leski 1977; Li et al. 2015).

In addition to race determination experiments, the examination of polymorphisms within well-characterized effectors has been important to the understanding of the coevolution of Cf resistance genes and effectors. Through alterations to effector genes, such as point mutations, deletions, and insertions of transposon-like elements, the pathogen can overcome Cf-mediated resistance, leading to the emergence of new races (Stergiopoulos et al. 2007b). Sequencing the previously characterized effector genes has enhanced our understanding of effector evolution and the ability of the pathogen to evade host resistance genes (Iida et al. 2015; Medina et al. 2015; Stergiopoulos et al. 2007a).

Because no teleomorph has been observed, P. fulva is hypothesized to reproduce asexually. Amplified fragment length polymorphism analysis of a global collection of isolates showed greater genotypic diversity than what would be expected for a primarily asexual fungus where only a few clonal groups exist (Stergiopoulos et al. 2007a). Mating type is another means to assess the possibility of sexual reproduction. Heterothallic fungi require opposite mating types to reproduce sexually, and for filamentous ascomycetes, mating type is often controlled by the MAT locus, containing the idiomorphs MAT1-1 and MAT1-2 (Turgeon 1998; Turgeon and Yoder 2000). Based on similarity to homologous genes in other ascomycetous fungi, MAT1-1-1 and MAT1-2-1 have been cloned and characterized in P. fulva (Stergiopoulos et al. 2007a). In studies characterizing 86 isolates from around the world and 133 isolates from Japan, both mating types were present in the populations, but departure from a 1:1 ratio of the mating types was observed, which did not support the hypothesis of random mating and sexual reproduction in P. fulva (Iida et al. 2015; Milgroom 2017; Stergiopoulos et al. 2007a). Uncertainty remains regarding the role of sexual reproduction in P. fulva.

Other questions remain. In particular, the genetic diversity and population structure of P. fulva populations in the United States are uncharacterized. An improved knowledge of the races, genetic diversity, and population genetic structure of P. fulva will clarify whether regional differentiation exists and form the basis to eventually provide more precise recommendations for the management of tomato leaf mold in the region. Thus, the first objective of this study was to determine the race structure of populations of P. fulva in the Northeastern United States and Minnesota. The second objective was to determine the genetic diversity and population structure among these isolates.

MATERIALS AND METHODS

Fungal isolation.

Samples of tomato leaves with leaf mold were collected in the summers of 2015 through 2019 from states in the Northeast United States and Minnesota. In total, 50 single-conidial isolates were collected, which included 15 isolates from Minnesota, and 35 additional isolates from the northeastern states of New York, New Hampshire, Vermont, and Massachusetts (Fig. 2; Supplementary Table S1). To isolate P. fulva, 5-mm leaf disks were surface-sterilized in 70% ethanol for 1 min before being rinsed in sterile water, placed in a 10% bleach solution for 1 min, and finally thoroughly rinsed with sterile water. The leaf disks were placed on sterile filter paper to air-dry for 2 min before they were placed on potato dextrose agar (PDA), amended with 1 ml of 90% lactic acid per liter of media. To obtain single-conidial isolates, a small agar plug from a sporulating culture was placed in 1 ml of deionized water in a 1.5-ml tube. After vortexing tubes for 5 s, 1 μl of the conidial suspension was added to a new tube with 999 μl of deionized water. One-hundred microliters of the 1:1,000 diluted solution was pipetted and spread on PDA using an L-shaped cell spreader. After 48 h, single germinating conidia, visible by stereo microscope, were transferred with a scalpel to new Petri dishes containing PDA. All cultures were incubated at 20 to 22°C in the laboratory. Ten small plugs from the resulting cultures were added to 1 ml of 30% glycerol stocks for long-term storage at −80°C.

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Identification of the causal fungus.

To confirm the identity of P. fulva, the internal transcribed spacer (ITS) region was amplified by PCR using the ITS4 and ITS5 primers (White et al. 1990). To extract DNA, mycelia were either collected directly from PDA using a sterilized scalpel, or from cultures of P. fulva grown in sterile potato dextrose broth which were vacuum-filtered, and the mycelia collected for DNA extraction. Twenty milligrams of mycelia was placed in 2-ml round-bottom tubes with two 3-mm stainless steel beads (Qiagen, Valencia, CA). Tubes were placed in a 2 × 24 TissueLyser adapter set (Qiagen) and stored at −80°C for at least 2 h, to aid in pulverizing the mycelia. Mycelia was ground in a Mixer Mill MM 400 (Retsch Inc., Newton, PA) at 30 Hz for 1 min. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen), according to the manufacturer’s instructions.

The ITS region PCR amplification comprised 12.5 μl of Emerald Amp GT 2× Master Mix (Takara Bio Inc, Shiga, Japan), 9 μl of deionized water, 0.2 μM of the forward and reverse primers ITS4 and ITS5 (White et al. 1990), and 50 ng of template DNA for a final reaction volume of 25 μl. The PCR was performed in a C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA). PCR conditions included a denaturation step of 94°C for 4 min, followed by 33 cycles of 94°C for 45 s, an annealing step at 57°C for 45 s, and an extension step at 72°C for 1 min. The final step was 72°C for 5 min. Amplicons were Sanger-sequenced at the Cornell Institute of Biotechnology (Ithaca, NY) and queried against the National Institute for Biotechnology Information (NCBI, Bethesda, MD) nucleotide database using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov; Altschul et al. 1990; White et al. 1990).

Two isolates of P. fulva (17036 and 17038, identified using the described methods) collected from grower’s high tunnels in 2017 were used to complete Koch’s postulates (Supplementary Table S1). Koch’s postulates were tested using 4-week-old tomato plants of the susceptible cultivars ‘Moneymaker’ and ‘BHN 589’ in 3.57-liter pots containing LM-3 All Purpose Mix (Lambert Peat Moss Inc., Rivière-Ouelle, Québec, Canada). An L-shaped cell spreader was used to scrape conidia from 2-week-old cultures from the surface of the PDA prepared into a slurry with deionized water. The slurry was filtered through cheesecloth, the concentration of the solution was adjusted to ∼2.5 × 10⁵ conidia/ml, and then was placed in Nalgene aerosol spray bottles (Thermo Fisher Scientific, Waltham, MA). The 4-week-old tomato plants were placed in a high tunnel at Cornell AgriTech (Geneva, NY), and all leaf surfaces were sprayed to runoff with the conidial suspension. After 2 weeks, symptoms of disease and signs of the pathogen appeared on the foliage, and the fungus was reisolated on PDA. A similar protocol was followed to test Koch’s postulates with other Cladosporium-like fungi that were isolated from symptomatic leaf surfaces.

Race determination assays.

Seed for a differential set containing tomato lines with resistance genes Cf-2, Cf-4, Cf-5, Cf-6, Cf-9, and Cf-4 and Cf-11 in combination, as well as a tomato line with no known resistance genes, was obtained from either the Charles M. Rick Tomato Genetics Center (University of California, Davis, CA), the Centre for Genetic Resources (Wageningen University and Research, Wageningen, The Netherlands), or from commercial sources (Table 1). To obtain enough seed for race determination, four tomato seedlings of each genotype were placed individually in 7.57-liter pots containing LM-3 All Purpose Mix. The tomato plants were staked and pruned until fruit production was underway. Tomatoes were fertilized each week with Chem-Gro Tomato Formula (4-18-38) fertilizer (Hydro-Gardens Inc., Colorado Springs, CO), based on the manufacturer’s recommendations, with calcium nitrate (15.5-0-0) applied at a rate of ∼0.3 g/liter, and magnesium sulfate Epsom salt applied at a rate 0.3 g/liter. Tomato plants were grown to maturity in a climate-controlled greenhouse with a 16-h photoperiod. Fruit was harvested at maturity. Seed was collected from the fruit, cleaned in a 50% HCl solution, rinsed with trisodium phosphate cleaner, and then dried and stored in a cold room until required for the race assays.

TABLE 1. The races of Passalora fulva identified in the Northeast of the United States and Minnesota from inoculating the isolates on a differential set of tomato accessions

Tomato accession numbera	Cultivar or background genotype	Cf resistance gene present	Number of isolates causing disease
Seed obtained commercially	Moneymaker	No Cf gene	50/50
LA3043	Moneymaker background	Cf-2	44/50
LA3045	Moneymaker background	Cf-4	0/50
LA3046	Moneymaker background	Cf-5	0/50
LA2448	Ontario 7818	Cf-6	0/50
LA3047	Moneymaker background	Cf-9	0/50
CGN18402	Ontario 7716	Cf-4, Cf-11	0/50

^a Accessions with the “LA” prefix were obtained from the Charles M. Rick Tomato Genetics Center (Davis, CA), and CGN18402 was obtained from the Centre for Genetic Resources (Wageningen, The Netherlands).

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Race assays were conducted during the summers of 2019 and 2020 at the Cornell AgriTech high tunnel. The tomato seeds were sown in 50-cell flats containing LM-1 Germination Mix (Lambert Peat Moss Inc., Rivière-Ouelle, Québec, Canada) and were grown in a greenhouse for 4 weeks. In preparation for transplanting, tomato plants were fertilized once a week, after the first true leaves expanded, using Miracle-Gro All Purpose Plant Food (24-8-16; The Scotts Miracle-Gro Company, Marysville, OH). Tomato plants were transplanted into 3.79-liter pots and placed in the high tunnel. Each differential set of tomato plants (Table 1) was placed 1 m apart from the other sets, across three rows that were spaced 1.25 m apart. Including control tomato plants, 22 sets of differential plants could fit in the high tunnel. Because of space limitations, experiments were conducted at 3-week intervals throughout the summer. Each isolate was randomly assigned to two differential sets at a time, and the position of genotypes within blocks was randomly assigned. Thus, there were two replicates of each P. fulva isolate × tomato differential genotype combination. To prepare inoculum, each isolate was taken fresh from long-term storage to avoid any negative effects of serial microbial transfers on isolate fitness. A suspension of the stored conidia in water was prepared and spread on PDA in Petri dishes using an L-shaped spreader and cultured for 4 weeks at room temperature (20 to 22°C). For each isolate, conidia were washed from the media surface with sterile water, and the concentration of the inoculum adjusted to 2.5 × 10⁵ conidia per ml. Tomato plants were spray-inoculated to runoff as previously described using a separate handheld misting bottle for each isolate. After inoculation, the plants were regularly watered until disease symptoms developed after ∼14 days. Spray bottles and other equipment were thoroughly disinfected in a bleach solution between experiments. Plants were rated for the presence or absence of tomato leaf mold symptoms 16 to 18 days post inoculation, to ensure ample disease development before the rating.

Sequencing of effector genes and PCR amplification of mating type idiomorphs.

The primers used for PCR amplification and sequencing were previously described and are listed in Supplementary Table S2 (Iida et al. 2015; Medina et al. 2015). To amplify and sequence the effector genes, the same DNA extraction and PCR preparation protocols were followed that were described for the ITS region. For amplification of Avr2, Avr4, Avr4e, and Avr9, the PCR conditions comprised a denaturation step at 94°C for 5 min, 35 cycles of 94°C for 30 s, an annealing step at 60°C for 30 s, and an extension step of 72°C for 1 min. The final step was 72°C for 7 min. Sequences were aligned to previously published reference sequences (Supplementary Table S3) using the tool MUSCLE v.3.8.425 (https://kbase.us/applist/apps/kb_muscle/MUSCLE_nuc/release; Edgar 2004) in Geneious Prime v.2020.2.2 (https://www.geneious.com; Biomatters Inc., San Diego, CA). We identified polymorphisms within the coding regions of genes based on the position of the start codon of the reference genes. The main reference sequences were from race-0 isolate ‘ELH’ from Argentina (Medina et al. 2015). We also compared sequence data to additional reference sequences belonging to race-4 or race-5 isolates (Supplementary Table S3; Joosten et al. 1997; Luderer et al. 2002; Van den Ackerveken et al. 1992; Westerink et al. 2004). Sequenced genes were translated, and the results were compared with protein predictions from reference sequences, obtained from the NCBI Protein database.

The PCR reaction mixtures for the amplification of mating type idiomorphs were as described above for the Avr genes. The PCR conditions were as described in Stergiopoulos et al. (2007b). The PCR products were analyzed by gel electrophoresis on a 1% agarose gel stained with GelRed (Biotium Inc., Fremont, CA) and visualized under UV light. An exact binomial test (two-tailed) for goodness-of-fit was used to determine whether observed mating type frequencies within populations deviated from a 1:1 ratio using the R Stats package v.4.1.0 R (https://stat.ethz.ch/R-manual/R-devel/library/stats/html/00Index.html; R Core Team 2020). The full data set and the clone-corrected dataset (which was compiled after genotyping-by-sequencing; GBS), were analyzed.

GBS.

GBS libraries were prepared and sequenced at the University of Wisconsin-Madison Biotechnology Center DNA Sequencing Core Facility (Madison, WI). Libraries were digested with ApeKI and sequenced on an Illumina NovaSeq6000 (https://www.illumina.com), generating 150 base-pair paired-end reads. Replicate samples were included in the plate (Supplementary Table S1). Genotypes were called with the program TASSEL 5 GBS v.2 pipeline (https://bitbucket.org/tasseladmin/tassel-5-source/wiki/Tassel5GBSv2Pipeline; Glaubitz et al. 2014), using the forward reads as input. Sequence tags were aligned to the C. fulvum v.1.0 genome, obtained from the Joint Genome Institute (de Wit et al. 2012; Ohm et al. 2012). We used R v.4.0.2 (R Core Team 2020), the R package vcfR v.1.11.0 (https://cran.r-project.org/web/packages/vcfR/index.html; Knaus and Grünwald 2017), and VCFtools v.0.1.16 (https://mybiosoftware.com/vcftools-program-package-designed-working-vcf-files.html; Danecek et al. 2011) to retain only high-quality, single nucleotide polymorphisms (SNPs). Using vcfR, variants were filtered when read depth was <5 and >100 (Knaus and Grünwald 2017). Samples were omitted if they had >55% missing data. Variants with >20% missing data were also omitted. Only biallelic and polymorphic sites were retained, and given that the fungus is haploid, heterozygous genotype calls were also censored. SNPs were retained only when the minor allele frequency was >0.05. When replicate samples were present, the replicates with the least missing data were retained.

Analysis of GBS data were conducted with R v.4.0.2 (R Core Team 2020), in reference to the Population Genetics and Genomics in R Primer (https://grunwaldlab.github.io/Population_Genetics_in_R/). The R packages vcfR (Knaus and Grünwald 2017) and ggplot2 (https://ggplot2.tidyverse.org/; Wickham 2016) were used in the processing and visualization of data. A maximum-likelihood tree was made with the tools IQ-TREE (http://www.iqtree.org/; Nguyen et al. 2015) and ModelFinder (http://www.iqtree.org/ModelFinder/; Kalyaanamoorthy et al. 2017). The model K3Pu+F (http://www.iqtree.org/doc/Substitution-Models) was chosen as the best-fit substitution model. Branch supports were obtained with the Shimodaira-Hasegawa approximate likelihood rate test (Guindon et al. 2010) and the ultrafast bootstrap approximation test (Hoang et al. 2018), with 1,000 bootstrap replicates. The tree was visualized using the programs APE (https://cran.r-project.org/web/packages/ape/ape.pdf; Paradis et al. 2004), ggplot2 (Wickham 2016), and ggtree (https://bioconductor.org/packages/devel/bioc/vignettes/ggtree/inst/doc/ggtree.html; Yu et al. 2017). A map illustrating the counties sampled was constructed using the R package Maps (https://cran.r-project.org/web/packages/maps/index.html; Becker and Wilks 2021).

Clonal groups were identified using pairwise identity-by-state (IBS), as described in Vogel et al. (2020) and Carlson et al. (2017). For all pairwise combinations of isolates, the proportion of alleles that were shared at the nonmissing sites were calculated. To differentiate potential clones, a threshold of 99% was set based on visualization of a histogram of the IBS matrix data. Isolates beyond the 99% threshold were compiled into clonal groups. With the clone-corrected dataset, principal component analysis (PCA) plots were constructed using the R package Adegenet (https://cran.r-project.org/web/packages/adegenet/index.html; Jombart 2008; Jombart and Ahmed 2011). Using the clone-corrected data, pairwise F_st (Weir and Cockerham 1984) was calculated using the R package Hierfstat (https://cran.r-project.org/web/packages/hierfstat/index.html; Goudet 2005). The analog to F_st, G_st (Hedrick 2005; Nei 1972, 1973; Knaus and Grünwald 2017) was calculated with the clone-corrected dataset. Linkage disequilibrium (LD) decay of the clone-corrected data were visualized using PopLDDecay v.3.41 (https://github.com/BGI-shenzhen/PopLDdecay; Zhang et al. 2019). Pairwise LD was measured by the coefficient of determination r². The bin2 parameter was set to 10,000 bp.

Data availability.

Raw demultiplexed FASTQ files have been deposited at the NCBI Sequence Read Archive (BioProject accession number PRJNA734954). Scripts are available on Github (https://github.com/masudermann/tomato_leaf_mold).

RESULTS

Fungal isolation and identification.

We isolated P. fulva from a total of 50 symptomatic tomato leaf samples (Fig. 2; Supplementary Table S1). Other fungal species were isolated from leaf samples that were only sterilized in 10% bleach, rather than both ethanol and bleach. The other species of fungi were morphologically distinct, grew rapidly on PDA, and were darker gray in color when compared with P. fulva. In contrast, P. fulva colonies were slow-growing, remained confined to a small section of the plate, and had a lighter olive-green color (Fig. 3). BLAST searches and phylogenetic analysis of the ITS results revealed that the other species of fungi isolated had greatest sequence similarity to several Cladosporium spp. including C. cladosporioides and C. pseudocladosporioides. In contrast, P. fulva, as confirmed by ITS sequencing and morphological differences, was the only consistently isolated fungal species from diseased samples that were thoroughly surface-sterilized in ethanol and 10% bleach.

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The pathogenicity of P. fulva was confirmed by fulfilling Koch’s postulates with isolates 17036 and 17038 (Supplementary Table S1). Disease symptoms and signs appeared 14 days post inoculation of tomato plants. The pathogen was reisolated on PDA. We could not complete Koch’s postulates with isolates that were classified as other Cladosporium spp., as no disease symptoms developed after inoculation with the fungi.

Race determination experiments.

All 50 P. fulva isolates caused disease on ‘Moneymaker’, which has no known resistance genes to P. fulva. None of the 50 isolates caused any disease symptoms on tomato accessions containing either Cf-4, Cf-5, Cf-6, Cf-9, or Cf-4 and Cf-11 in combination. Forty-four isolates caused disease symptoms on tomato plants with the resistance gene Cf-2, whereas five isolates from New York (17052, 17053, 17057, 18013, and 19006) and one isolate (19008) from Massachusetts caused disease symptoms on ‘Moneymaker’ only (Table 1). Therefore, based on previous race nomenclature conventions, 44 isolates collected across the Northeast and Minnesota belonged to race 2, while six isolates belonged to race 0.

Analysis of allelic variation of four effectors.

Four effector genes, Avr2, Avr4, Avr4e, and Avr9, were sequenced for each of the 50 isolates to identify polymorphisms (Table 2). For Avr2, six polymorphisms were identified within the coding region, and one polymorphism was identified downstream of the coding region of the gene. The deletions and insertion result in frameshift mutations. The three Vermont isolates had a large deletion beginning at position 37, resulting in a truncated protein beginning after L12. Of the six race-0 isolates, all but one isolate had an adenine and guanine deletion downstream of the coding region (Tables 2 and 3). Finally, seven isolates, belonging to race 2 (17035, 17046, 17049, 18009, 19001, 19013, and 19017), did not produce a PCR amplicon with the Avr2 primers used (Medina et al. 2015). In Avr2, no isolate had more than one polymorphism relative to the reference sequences. There were 12 race-2 isolates from Minnesota and two isolates from Ontario County, New York that did not show any polymorphisms compared with one another or to the reference sequences (Tables 2 and 3; Supplementary Table S3).

TABLE 2. Genetic characteristics of isolates of Passalora fulva collected in the Northeast of the United States and Minnesota with information on the genotypes (clonal groups), the mating type gene that was PCR-amplified, and the race, location of isolation, and effector gene polymorphisms in the coding region for each isolate^a

Clonal group	Sample name	State	MAT-type	Race	Avr2	Avr4	Avr4e	Avr9
1	pf76	MN	MAT1-2-1	Race 2	No changes	No changes	c.244T>C, c.248T>C	c.23T>C
	pf77
	pf78
	pf79
	pf81
	pf90
	pf91
	pf93
2	17035	NY	MAT1-1-1	Race 2	Not amplified	g.128G>A	No changes	No changes
	17046
	17049
	18009
	19001
	19013	NH
	19017	NY
3	17036	NY	MAT1-2-1	Race 2	c.64delA	No changes	No changes	No changes
	17037
	17038
	19016
	19018
4	pf85	MN	MAT1-2-1	Race 2	No changes	No changes	No changes	c.23T>C
	pf88
	pf89
	pf92
5	19010	NH	MAT1-2-1	Race 2	c.64delA	g.128G>A	No changes	No changes
	19011
	19012
	19014
6	17052	NY	MAT1-2-1	Race 0	g.348_349delAG	No changes	No changes	No changes
	17053
	19008
7	17040	NY	MAT1-2-1	Race 2	c.64delA	No changes	No changes	No changes
	18019
	18024
8	19002	VT	MAT1-2-1	Race 2	Large deletion	g.128G>A	No changes	No changes
	19003
	19004
9	17043	NY	MAT1-2-1	Race 2	c.64delA	g.128G>A	No changes	No changes
	19009
10	16139	NY	MAT1-1-1	Race 2	No changes	No changes	c.244T>C, c.248T>C	c.23T>C
	18025
11	pf80	MN	MAT1-1-1	Race 2	c.121_122delTA	No changes	c.244T>C, c.248T>C	c.23T>C
	pf82
12	19006	MA	MAT1-1-1	Race 0	g.348_349delAG	No changes	No changes	No changes
13	18010	NY	MAT1-1-1	Race 2	c.209insA	g.128G>A	No changes	No changes
14	18012	NH	MAT1-1-1	Race 2	c.2T>C	No changes	c.244T>C, c.248T>C	No changes
15	18013	NY	MAT1-1-1	Race 0	g.348_349delAG	No changes	No changes	No changes
16	17057	NY	MAT1-2-1	Race 0	No changes	g.128G>A	No changes	No changes
17	18005	MA	MAT1-2-1	Race 2	c.46delG	No changes	c.244T>C, c.248T>C	No changes
18	pf84	MN	MAT1-2-1	Race 2	c.2T>C	No changes	c.244T>C, c.248T>C	No changes

^a Minnesota (MN), New York (NY), New Hampshire (NH), Minnesota (MN), Vermont (VT), and Massachusetts (MA).

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TABLE 3. The polymorphisms and predicted effects on proteins for four effector genes in 50 isolates of Passalora fulva collected in the Northeast of the United States and Minnesota in reference to the race-0 isolate “ELH” defined by Medina et al. (2015), as well as additional race-4 and race-5 isolates^a

Gene	Length of reference sequence	Number of polymorphisms		Polymorphisms in coding region and predicted effects on proteins
		Coding region	Noncoding region	Number of isolates with polymorphisms	Polymorphism	Type of polymorphism	Predicted effect on protein
Avr2	562	5	1	2	c.2T>C	SNP	M1T-nonsynonymous substitution
				1	c.46delG	Indel	V17fs-frameshift mutation
				14	c.64delA	Indel	K23fs-frameshift mutation
				2	c.121_122delTA	Indel	Y41fs-frameshift mutation
				1	c.209insA	Indel	H70fs-frameshift mutation
				5	g.348_349delAG	Indel	No effect
				3	Large deletion starting at c.37	Indel	Truncated protein beginning after L12
Avr4	785	0	1	18	g.128G>A	SNP	No effect
Avr4e	609	2	0	15	c.244T>C	SNP	F82L-nonsynonymous substitution
				15	c.278T>C	SNP	M93T-nonsynonymous substitution
Avr9	675	1	0	16	c.23T>C	SNP	V8A-nonsynonymous substitution

^a Sample isolates were collected in the Northeast of the United States and Minnesota; SNP, single nucleotide polymorphism.

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Only one SNP was identified upstream of the coding region of Avr4. The nonreference allele was present in 18 isolates from the Northeastern states (Table 3). Relative to the race-0 reference strain (Medina et al. 2015), but not the Avr4e reference sequence (Westerink et al. 2004), two SNPs were observed associated with Avr4e. The same 15 isolates contained the nonreference alleles, resulting in two potential nonsynonymous substitutions (Table 3; Supplementary Table S3). Finally, only one SNP was identified within the coding region of Avr9, and 16 isolates contained the nonreference allele. This corresponded to a nonsynonymous substitution (Tables 2 and 3).

Isolates that belonged to the same clonal group and had the same genotype had at least one polymorphism compared with the reference sequences within one or more of the effector genes. If a clonal group did have a polymorphism, most often isolates within the group had only one polymorphism for a given gene, relative to reference isolates.

Mating type characterization.

We were able to amplify only one, but not both, MAT genes in all isolates (Table 4). Overall, the MAT1-1-1 gene was amplified in 15 of the 50 isolates (30% of the population), and the MAT1-2-1 gene was amplified in the remaining 35 isolates (70%). A two-tailed exact binomial test for goodness-of-fit indicated a mating type frequency that deviated from an equal ratio of mating type alleles (P = 0.007; Table 4). Analysis of the clone-corrected data indicated that the mating type genes were in equilibrium (P = 0.48).

TABLE 4. Mating type gene frequencies for 50 isolates of Passalora fulva collected from New York (NY), Minnesota (MN), Vermont (VT), New Hampshire (NH), and Massachusetts (MA)

State/Sample	Sample size	Mating type gene		Pa
		MAT1-1-1	MAT1-2-1
NY	24	10 (41.7%)	14 (58.3%)	0.54
MN	15	2 (13.3%)	13 (86.7%)	0.007
VT	3	0 (0%)	3 (100%)	N/A
NH	6	2 (33.3%)	4 (66.7%)	0.69
MA	2	1 (50%)	1 (50%)	N/A
Total	50	15 (30%)	35 (70%)	0.007
Total (clone corrected)	18	7 (39%)	11 (61%)	0.48

^a Probabilities were calculated using a two-tailed exact binomial test, to test whether mating type frequencies deviated from ratios of 1:1. Bold values indicate a significant deviation from a 1:1 ratio. When N/A is listed, probabilities were not calculated because sample sizes were <5.

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Considering race, two of the race-0 isolates had the MAT1-1-1 gene (33.3%), and four of the race-0 isolates had the MAT1-2-1 gene (66.7%). For the race-2 isolates, 13 of the 44 (29.5%) had the MAT1-1-1 gene, and 31 of the 44 isolates (70.5%) had the MAT1-2-1 gene. For the race-2, but not race-0 isolates, a two-tailed exact binomial test for goodness-of-fit indicted a mating type frequency that deviated from an equal ratio of mating type alleles (P = 0.01).

GBS.

Of the 34,044 variants discovered in the raw dataset, 7,514 SNPs remained in the filtered dataset. Mean read depth per individual was 23.56 and the mean depth per site averaged across individuals was 23.13.

To better understand the population structure of isolates in this study, a maximum-likelihood tree was constructed with 1,000 bootstrap replicates (Fig. 4). In many instances, isolates clustered together by state, including into several monophyletic groups. Many isolates from the same region and county grouped together, including isolates from the same county that were collected in different years. This could be indicative of the pathogen over-wintering.

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The isolates from the same counties that were collected in different years included 17040 (collected in 2017) and 18019 (collected in 2018), from Essex County, NY; the isolates 16139 (collected in 2016) and 18025 (collected in 2018) from Ontario County, NY; and many Minnesota isolates collected in Anoka County in 2016 compared with Pf91, which was collected in 2017 (Fig. 4; Table 2; Supplementary Table S1).

There were also examples of clades containing isolates from multiple states. For example, one clade contained isolates from MN (Pf84) and NY (18005). Another exception was an isolate from New Hampshire (19013). It grouped more closely to New York isolates than to isolates from New Hampshire (Fig. 4).

An IBS similarity matrix revealed that the mean pairwise IBS between isolates was 0.715. The mean IBS between replicate samples was 0.99994, corresponding to an error rate of 0.006%. The lowest IBS between a pair of replicate samples (Pf79 and Pf79-2) was 0.99968, corresponding to an error rate of 0.032% (Supplementary Table S4). A histogram of the pairwise IBS matrix showed that IBS between most isolates ranged between 0.6 and 0.8, with a separate, strong peak at ∼0.99. Given that the peak was distinct from the distribution of the majority of pairwise IBS values, and aligned with the IBS identified between replicate samples, we considered any group of isolates featuring pairwise IBS > 0.99 to represent the same unique genotype and comprise a clonal group.

In total, 18 clonal groups with distinct genotypes were identified (Table 2). Each of the 11 clonal groups with more than one isolate was comprised of either isolates from Minnesota or the Northeast, but no clonal groups were comprised of isolates from both geographic regions. There were seven genotypes that were represented by the single isolates 17057, 18005, 18010, 18012, 18013, and 19006, and the Minnesota isolate Pf84. The largest clonal group was comprised of eight isolates from Minnesota collected in 2016 and 2017. The second largest clonal group was comprised of six isolates from New York and one isolate from New Hampshire, collected between 2017 and 2019 (Table 2). Monophyletic groups within the maximum-likelihood tree are comprised of isolates from the same clonal group (Fig. 4; Table 2). All isolates within a clonal group had the same mating type. Isolates in seven of 18 (39%) groups had MAT1-1-1 and 11 of 18 (61%) groups had MAT1-2-1. A two-tailed exact binomial test for goodness-of-fit does not suggest a mating type frequency that deviates from an equal ratio of mating type alleles (P = 0.48; Table 4).

The PCA indicated 22% of the variance in the SNP genotype data was explained by principal component (PC) 1, which largely differentiated Minnesota isolates from the isolates collected in New York and the other Northeastern states. Exceptions were the two New York isolates 16139 and 18025 that belonged to clonal group 10 (Table 2). The point depicting clonal group 10 was adjacent to several of the clonal groups comprised of Minnesota isolates, a result also observed in the maximum-likelihood tree (Fig. 4). PC 2 explained 15% of the variance and differentiated New Hampshire isolates from the rest of the isolates (Fig. 4).

The F_st value between New York and Minnesota isolates was 0.14 and the G_st value was 0.132, suggesting moderate differentiation between the isolate groups. Considering the small sample size of the Vermont, New Hampshire, and Massachusetts isolates, only the comparison between the New York and Minnesota populations was examined.

The LD is presented in a plot showing pairwise r² values between SNPs as a function of distance, averaged in 10-kb bins (Fig. 5). LD decayed with greater distance between sites on the chromosomes. Whereas the mean r² between sites within 50 kb of each other was equal to 0.286, sites between 250 and 300 kb of each other had a mean r² = 0.162.

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DISCUSSION

The tomato leaf mold pathogen P. fulva was isolated from symptomatic tomato plants, as confirmed by sequencing the ITS region and demonstration of Koch’s postulates. Other Cladosporium spp. belonging to the C. cladosporioides complex (Bensch et al. 2010; Crous 2009; Crous et al. 2007, 2009; Schubert et al. 2007; Zalar et al. 2007) were isolated from symptomatic tomato plants, but appeared to be secondary colonizers, as they did not cause symptoms on tomato plants. For samples collected from growers’ high tunnels, double surface sterilization with 70% ethanol and 10% bleach was the only way to successfully isolate P. fulva and prevent growth of the secondary fungi.

In Argentina, in addition to P. fulva, two Cladosporium species, C. sphaerospermum and C. cladosporioides, were isolated from symptomatic tomato plants (Medina et al. 2015). As with our observations, the isolates from Argentina were found to be morphologically different from P. fulva and grew rapidly in culture (Medina et al. 2015). The Cladosporium genus is diverse, yet few reports describe pathogenicity of different species on tomato plants. Some Cladosporium species, including C. oxysporum and C. cladosporioides, have been reported to cause a tomato leaf spot (Huang et al. 2013; Lamboy and Dillard 1997; Robles-Yerena et al. 2019). Other research has suggested that C. cladosporioides can cause black mold on postharvest tomato fruit (Ma et al. 2020). The results of our evaluation suggests that P. fulva is the cause of tomato leaf mold, while the Cladosporium species isolated were secondary colonizers.

Most of the 50 isolates were race 2; only six isolates were race 0. Isolates from the same clonal group belonged to the same race. Our results are congruent with the findings from race determination experiments in Argentina (Medina et al. 2015). This contrasts with the race structure in other locations, including Japan, China, and Europe, where isolates that overcome Cf-2, Cf-4, Cf-5,and Cf-9 have been identified. Isolates have also overcome Cf-11 in Japan and Europe (Iida et al. 2015; Li et al. 2015; Lindhout et al. 1989; Yoshida et al. 2021). In Japan, the defeated R genes were gradually introduced into tomato lines beginning in the 1960s (Iida et al. 2015; Yoshida et al. 2021). In Argentina, because tomato leaf mold was a relatively new disease in the mid-2010s, it was concluded that the limited race structure of P. fulva was the result of tomato cultivars not containing as many Cf resistance genes compared with the cultivars grown in Europe, Japan, and China, leading to a less variable race structure (Iida et al. 2015; Li et al. 2015; Lindhout et al. 1989; Medina et al. 2015). It is possible that a similar scenario exists in the United States, where tomato leaf mold is a re-emerging disease in tomato cultivars commonly grown in high tunnel and greenhouse environments. A large body of literature exists from the 1930s to the 1970s describing research on tomato leaf mold resistance breeding efforts and corresponding race determination studies that took place most often in Canada, but also in the United States (Bailey 1950; Langford 1937). While the race nomenclature gradually shifted and understandings of gene-for-gene interactions between the pathogen effectors and tomato Cf genes became more complete, the studies demonstrated that several predominant races emerged, directly corresponding to the introgressed resistance genes in tomato cultivars (Kerr and Bailey 1966). The early studies from locations in Canada identified races in addition to the two we identified (races 0 and 2; Bailey 1950; Kerr and Bailey 1966; Langford 1937). The loss of pathogen diversity over the past 90 years could be because of changes in host genotypes cultivated for tomato production in the United States and Canada.

Despite the small number of races we identified, most of the isolates from Minnesota and the Northeast overcame Cf-2−mediated resistance. We know that Cf-2 resistance was introgressed from chromosome 6 of the wild species L. pimpinellifolium (Rivas and Thomas 2005). In Japan, Cf-2 was incorporated into commercial lines in the 1960s, and within a decade, race-2 isolates were identified (Iida et al. 2015). Given that 44 out of 50 isolates in our study overcame resistance to Cf-2, growers are advised to avoid relying on tomatoes with the Cf-2 resistance gene.

Mutations in effectors, including indels and SNPs, are an important way in which fungal pathogens evade recognition by host resistance proteins and facilitate virulence (De Wit et al. 2009; Selin et al. 2016). When compared with previous studies that examined allelic variation of effector genes within P. fulva, we observed less allelic variation within the four effector genes Avr2, Avr4, Avr4e, and Avr9 (Iida et al. 2015; Stergiopoulos et al. 2007b). Isolates from a given clonal group shared polymorphisms and patterns of allelic variation. Polymorphisms were more often seen between isolates from the same geographic location than the year of collection, suggesting that location plays an important role in the variation of effector genes within the pathogen, potentially because of on-farm overwintering of the pathogen. Thus, care should be taken to sanitize high tunnel and greenhouse environments and remove plants and plant debris at the end of the season.

Most of the polymorphisms in Avr2 were predicted to result in frameshift mutations that prevent the full translation of the Avr2 protein, and this parallels previous observations (Luderer et al. 2002; Stergiopoulos et al. 2007a). When Avr2 binds to the tomato extracellular cysteine protease Rcr3, a hypersensitive response by Cf-2 is triggered (Luderer et al. 2002; Rooney et al. 2005). As we would expect, only the race-2 isolates contained polymorphisms within the coding region, suggesting the polymorphisms resulted in protein modifications or deletions that ultimately resulted in a lack of recognition by Cf-2, which led to disease. In addition to the smaller indels observed in some isolates, a substantial deletion was found in Vermont isolates 19002, 19003, and 19004, which spanned across both the coding and noncoding region and was predicted to result in a truncated Avr2 protein. These three isolates were collected from ‘Berkeley tie dye’ on the same farm in Addison County, Vermont in July of 2019 and belonged to the same clonal group.

There were seven race-2 isolates for which an amplicon was not produced after PCR amplification using Avr2 primers; these belonged to clonal group 2 and were from New York and New Hampshire. It is possible that the Avr2 gene, or a portion of the gene, was deleted in these isolates, preventing PCR amplification. Further investigation is also needed with regard to the 12 isolates from Minnesota and two isolates from Ontario County, New York that belonged to race 2, yet did not show any polymorphisms compared with the race-0 reference sequence (Table 2). Additional analysis of these isolates, such as examination of gene expression, is warranted beyond simply comparing gene and predicted protein sequences.

Within the other effector genes, SNPs were observed more frequently. For Avr4e and Avr9, SNPs resulting in nonsynonymous substitutions were observed, rather than deletions causing frameshifts. In Avr4e, the predicted substitutions were p.F82L and p.M93T. Based on previous research, these two nonsynonymous substitutions allow P. fulva to overcome Hcr9-4e (also known as Cf-4e)-mediated resistance (Westerink et al. 2004). We did not include Hcr9-4e in our differential set, so this result was not experimentally confirmed. The same nonsynonymous substitutions were also noted in other studies (Iida et al. 2015; Stergiopoulos et al. 2007b).

With regard to Avr9, the one SNP we identified that resulted in the nonsynonymous substitution p.V8A, was observed in previous studies of allelic variation in a world-wide collection of isolates and in Japan (Iida et al. 2015; Stergiopoulos et al. 2007b). The effect of this nonsynonymous mutation is unknown. Functional experiments are needed to understand the effect that the polymorphism might have on protein structure and function. Finally, there were no polymorphisms in the coding region of Avr4, which might suggest that there is less selection on the pathogen to overcome Cf-4-mediated resistance in contrast to Avr2, which appears to be under the greatest selection.

P. fulva is known only by its asexual stage. Therefore, it is assumed that the pathogen population is clonal, and any genetic variation is because of mutation, in the absence of recombination, as demonstrated by the change in race structure in response to the deployment of tomato resistance genes (Joosten and De Wit 1999; Stergiopoulos et al. 2007b; Westerink et al. 2004). The amplification of mating type idiomorphs showed that 30% of our isolates had the MAT1-1 idiomorph, while 70% had MAT1-2. Interestingly, only the full dataset showed a significant deviation from a 1:1 mating type ratio.

Previously, the MAT1-1 mating type was observed at a higher frequency in a global collection of isolates (61%), as well the subset of isolates from Europe (64%; Stergiopoulos et al. 2007b), whereas in Japan, both mating types were present, with a bias for the MAT1-2 (73%) mating type (Iida et al. 2015). Mating type genes have also been cloned in the ascomycete fungi Fusarium oxysporum and Alternaria alternata (Arie et al. 2000). Like P. fulvum, sexual reproduction has not been observed in these species. There is some evidence to suggest that A. alternata exhibits cryptic sex based on the observations of 700 fungal isolates from potato (Meng et al. 2015). Considering that our sample size was small (50 isolates), a larger sample size in all subpopulations in each year would be helpful to better understand the frequency of mating types, and reproductive strategies.

Our clone-corrected dataset showed greater genotypic diversity than we hypothesized. Previous amplified fragment length polymorphism analysis also pointed to higher-than-expected genotypic diversity (Stergiopoulos et al. 2007b). Among 50 isolates, we identified 18 clonal groups with unique genotypes, including 11 genotypes with more than one individual. Besides one exception, isolates within the same clonal group were also from the same geographic location. In one instance in New Hampshire, isolate 19013 had the same genotype as New York isolates in clonal group 2.

Isolates from the same region, regardless of year of isolation, often grouped together in monophyletic groups. Interestingly, all the Vermont isolates grouped together into a single monophyletic group, while the isolates from all the other states belonged to multiple clades. Even though all the isolates belonged to races 0 or 2, isolates from both races could have either mating type idiomorph. Other population studies on P. fulva have similarly described isolates from the same race having different mating types (Iida et al. 2015; Stergiopoulos et al. 2007b). As visualized in the maximum-likelihood tree, PCA plots, and F_st and G_st analyses, isolates collected in Minnesota were genetically differentiated compared with locations in the Northeast. The differences between the two populations suggests the pathogen is not being dispersed large distances, which may be why the Minnesota and Northeast isolates remain genotypically distinct. Tomato leaf mold is often not observed until midseason, and it is unlikely that the disease spreads on transplants, which would be one potential method for longer-distance dispersal of genotypes. Additional sampling and analysis will provide confirmation of the regional differentiation between populations from the Northeast and Minnesota.

There was a gradual decay in LD with increasing distance between sites on the chromosomes, which suggests that genetic recombination takes place in P. fulva populations. The existence of genetic recombination is also consistent with the large number of clonal groups. A larger sample size is necessary to better understand the extent to which genetic recombination occurs in P. fulva. Evidence of genetic recombination does not necessarily indicate the presence of a sexual cycle in P. fulva, but could result instead from parasexuality leading to mitotic crossing-over (Pontecorvo 1956). In P. fulva, a parasexual cycle has been induced but never observed in the field (Stergiopoulos et al. 2007b). Nevertheless, the consistent identification of both mating type alleles in pathogen populations, and evidence of genetic recombination, raises the possibility of reproductive modes in addition to asexual spore production.

The objectives of this study were to determine the race structure and genetic diversity of isolates of P. fulva collected in the Northeastern United States and Minnesota. We observed that isolates from the two regions belong primarily to race 2, with a small number belonging to race 0. Planting tomato cultivars with a resistance gene in addition to Cf-2 will aid in the management of tomato leaf mold. Moderate regional differentiation exists between isolates collected in Minnesota compared with the isolates collected in New York and neighboring states. Both mating type genes were PCR-amplified in the isolate collection. While only two races were identified, 18 different clonal groups were confirmed, suggesting greater genetic diversity between isolates than might be expected from a strictly asexual fungus.

ACKNOWLEDGMENTS

We thank Holly Lange, Rachel Kreis, and Andrew Aldcroft for assistance isolating fungi from leaf samples, and for assistance fulfilling Koch’s postulates; Cornell Cooperative Extension educators including Amy Ivy and Judson Reid for assistance in collecting tomato leaf samples; extension specialists in New Hampshire, Vermont, and Massachusetts for mailing us leaf mold samples; and Colin Day and Garrett Giles for their assistance with the preparation, planting, and clean-up of the high tunnel race determination experiments.