View Tables and Figures in the PDF version of the 2023 CucCAP Squash Team Report.
Team Members:
- Florida: Geoffrey Meru, Yuqing Fu, Prerna Sabharwal, Swati Shrestha and Shailesh Acharya
- Michigan: Mary Hausbeck, Carmen Medina-Mora, David Perla, Matthew Uebbing
- New York Michael Mazourek, Chris Smart, Colin Day, Libby Indermaur, Gregor Inzinna, Taylere Herrmann
- Puerto Rico: Angela Linares
Map and develop markers for disease resistance
QTL mapping of resistances | Powdery Mildew – C. pepo
To date, no natural source of powdery mildew resistance has been reported in C. pepo. Although a single gene resistance (designated PM-0) has been described in a wild Cucurbita species (C. okechobeensis), its sole utilization in current cultivars increases the chance of resistance breakdown which could lead to yield loss. We evaluated 207 USDA germplasm accessions of C. pepo for PM resistance under greenhouse conditions at Cornell University (CU) and under field conditions at Michigan State University (MSU). Seeds of each accession (n=15) were grown in three replicates in a randomized complete block design. Success PM (carrying PM-0) and Early Prolific Straightneck cultivars were used as resistant and susceptible checks, respectively. Pathogen inoculum was provided through naturally infected plants. At the 6th true-leaf stage, symptom severity data were collected on a scale of 0-100% and 0-5 at CU and MSU, respectively, based on visible pathogen sporulation on the surface of top 4th leaf, bottom 4th leaf, stem above 4th leaf, and stem below 4th leaf. For CU, overall mean disease severity was highest in top 4th leaf (88.5%), followed by stem below 4th leaf (22.3%), bottom 4th leaf (9%), and least in stem above 4th leaf (6.5%). For MSU, the bottom 4th leaf had the highest disease severity (40%) followed by stem above the 4th leaf (28%). Success PM (R) showed consistent resistance across reps, with a mean disease severity of 0% in the stem below 4th leaf. However, Early Prolific (S) was susceptible with mean disease severity of 20% in stem below 4th leaf. Data from CU showed that accessions 189, 55, 70, 48, 205, 80, 144, 107 and 71 were the most resistant and accessions 22, 7, 120, 108, 104, 92, 99 and 56 were the most susceptible. Accession 189 showed the highest resistance in both CU and MSU datasets, with a mean disease severity of 7% in the stem below the 4th leaf. Further evaluation of the germplasm will be continued in FL and NY in 2023, and the data will be used to conduct GWAS to identify novel resistance alleles for summer squash breeding.
QTL mapping of resistances | Powdery Mildew – C. moschata
A natural source of resistance has been identified in C. moschata. In contrast to the wild species derived resistance, PM-0, that was mapped in CucCAP 1, this resistance is recessive and protects the upper leaf surface. Powdery mildew resistant and susceptible bulks (n = 22 per bulk) of the
F2 along with the parents were sequenced using NextSeq 500/550 and reads analyzed with the
QTL-seq bioconda package. The qtl-seq delta SNP index output indicated the novel PM resistance lies in a 2 Mb region of the genome. Using PCR markers developed from the vcf file output of the snpEff package, we scanned the region for SNP calls associated with the resistant and susceptible parents and the resistant and susceptible bulks. We have created 11 PCR markers spread across a now smaller 1.3 Mb region and have seven that can differentiate resistant from susceptible individuals; we were able to shrink the region down to 1 Mb based on three individuals in the resistant bulk that have crossovers on the downstream side and three resistant and one susceptible individual that have crossovers on the upstream side of the region.
QTL mapping of resistances | Phytophthora – C. pepo
Resistance to Phytophthora crown rot in UF breeding line #394-1-27-12 is controlled by three complementary dominant genes (R1R2R3). Introgression of these loci into C. pepo background is needed to complement the low-moderate level of resistance available in this species. To facilitate interspecific resistance transfer, an RNA-seq study was conducted to identify potential causal R genes that would facilitate the development of functional markers for marker-assisted selection. Breeding line #394-1-27-12 (R) and Butterbush (S) were challenged with a virulent isolate of P. capsici, and crown tissues were sampled at 0, 12, 24, 72 and 120 hours post infection (hpi) with three technical and three biological replicates for each time point. On average 90% of the genes were uniquely mapped to C. moschata var. Rifu reference genome. Transcript data for #394-1-27-12 and Butterbush showed strong correlation among replicates (85%). Overall, there were 4,377 differentially expressed genes (DEG) across the two genotypes, of which 1,116 and 613 were upregulated or downregulated in #394-1-27-12, respectively. The DEG were highest at 72 and 120 hpi, but lowest at 24 hpi. Co-expression analysis revealed that 213 and 48 genes overlapped in expression at 72 and 120 hpi in #394-1-27-12, respectively. Gene ontology and pathway enrichment analysis revealed that most of the upregulated genes were involved in ‘oxidation-reduction’, ‘response to stress’ and ‘response to wounding’ networks. Among these, ten genes involved in ‘stress / redox’ response were co-expressed at 72 and 120 hpi, including defensin-like proteins, respiratory burst proteins, peroxidases, MYB transcription factors, and cytochrome P450 protein. Interestingly, several genes, including CmoCh04G003840 (MYB transcription factors), CmoCh04G001860 (Subtilisin-like serine endopeptidase) and CmoCh11G009740/ CmoCh11G009720 (Germin-like proteins) were within confidence intervals of previously resistance QTLs in #394-1-27-12. Functional characterization of these genes will determine their role in disease resistance against Phytophthora crown rot in UF breeding line #394-1-27-12.
QTL mapping of resistances | Phytophthora – C. maxima
The Hausbeck Lab (MSU) (Section 3) identified cultivars that are promising for native Phytophthora capsici resistance in C. maxima. The Mazourek group has made F2 populations from crosses between the susceptible, commonly grown cultivar Golden Delicious and these other cultivars with promising resistance. Future work will focus on seedling screens for crown and root rot resistance and genotyping for mapping.
Marker development and verification | Powdery Mildew – C. moschata
For recessive mildew resistance in C. moschata, one marker in the middle of the 1Mb region harboring the resistance was found to contain a 9bp insertion in the resistant individual and can be used as a polymorphic PCR marker that can be visualized during gel electrophoresis. This should help us to identify resistant individuals more quickly and cost-effectively. This spring we plan to run the indel marker on 800 F2s and then run flanking markers on those individuals homozygous for the resistant parent allele for the indel marker. Individuals that have crossovers will be selected to go to the field and their resistant genotype and phenotype verified and the haplotype through this region will be explored through PCR markers and resequencing as necessary.
Introgress, pyramid/stack resistances into advanced breeding lines | powdery mildew – C. pepo, C. moschata
Breeding lines for processing squash
Two field trials were conducted to evaluate resistance to CPM in processing squash breeding lines in 2022 in NY. One trial evaluated CPM resistance in improved ‘Dickinson’ lines. ‘Dickinson’ is a large fruited industry standard processing cultivar that is related to butternut squash and susceptible to CPM. The two parents ‘Bugle’ (butternut, CPM resistant) and ‘Dickinson’ (CPM susceptible) were assessed alongside four of the most promising progeny from the breeding lines generated by this original cross. Each of the four progeny contains two copies of the CPM resistance gene Pm-0. Of the four progeny tested, Accession 3 had significanty less disease than ‘ Dickinson’ and was no different from ‘Bugle’ (Table 1). This demonstrates that CPM resistance has been bred into a commercially desirable variety. The number of fruit did not significantly differ across the progeny and ‘Dickinson’ (Table 1); ‘Bugle’ had significantly more marketable fruit per ten-plant plot, and the fruit are smaller, which is normal and expected for this genotype. Accession 3 had internal fruit and processing qualities that resemble ‘Dickinson’ in canning trials conducted in the fall of 2022. Further evaluations of Accession 3 are planned for the 2023 field season.
The second trial included 15 accessions of Cucurbita maxima, representing multiple market classes, to search for genetic resistance that could be bred into more susceptible cultivars. The squash accessions ‘Tabalque’, ‘Zapallito e Tronco’, an ‘Plomo Ruso’ had numerically the least disease, but did not have statistically less disease than any other variety except ‘Mayo Blusher’ (Table 2, Figure 2). Currently, C. maxima cultivars do not carry the Pm-0 gene that is common in butternuts (C. moschata) and are generally susceptible to CPM. Breeders are working towards incorporating resistance into commercially desirable C. maxima varieties.
Fruit processing
A protocol was developed in consultation with Olga Padilla-Zakour (Cornell University Department of Food Science) to process fruit at the lab-scale. For each accession in both ‘Dickinson’ and C. maxima trials, subsets of representative fruit were cooked for 30 minutes in a 3-tier, 9 qt electric steamer. Cooked fruit was then processed in a Victoria Food Mill and Sauce Maker (Model VKP250) using the pumpkin screen. Cheese cloth was used to press out 10% of free water by weight for products with excess moisture. Accessions that required water removal are noted in Tables 3 and 4. Final products were canned in triplicate using 4 oz Ball Mason jars in a 6 qt Instant Pot Max under high pressure for 115 minutes. Excess products were frozen for later pressure canning with an industrial retort. Processing was completed between 10/19 – 10/28/22. Sections of raw fruit weighing approximately 20 g were lyophilized in triplicate to estimate dry matter. Jars were opened on 1/11/23 to assess water separation, measured as the weight (kg) of free water that strained from each jar after 30 seconds. Because each accession was only processed and canned once, canning yield data is not replicated.
Among ‘Dickinson’ breeding lines, dry matter in fruit from each progeny was no different from ‘Dickinson’ (Table 3). ‘Bugle’ fruit was significantly drier, with no water separation and dry matter comprising 18.40% of its fresh weight, compared to 9.38% in Accession 3. Canning yield ranged from 36.68% (Accession 6) to 49.48% (Accession 3) of fresh weight. Accessions 3, 4, and 5 had significantly less free water separate compared to ‘Dickinson’, while Accession 6 was no different from Accessions 3, 4, and 5, and ‘Dickinson’. Overall, all progeny performed comparably to ‘Dickinson’ but not ‘Bugle’, which is promising for their advancement.
Fruit quality differed substantially within C. maxima accessions (Table 5). ‘Kestane’ fruit was among the moistest, with dry matter comprising only 5.22% of its fresh weight, and was no different from 9 other accessions with dry matter below 14.44%. Fruit from ‘Thunder F1’ were the driest; interestingly, this was among the most challenging to cut and prepare for processing. Canning yield spanned large numeric differences, from 34.62% (‘Kestane’) to 69.61% (‘Zapallito de Tronco’) of fresh weight. Seven accessions had no water separation. ‘Buttercup Burgess Strain’, ‘Amzibegovska’, and ‘Golden Delicious’ had significantly less free water separate compared to ‘Plomo ruso’, No. 7488, ‘Kestane’, and ‘Zapallito de Tronco’. ‘Dickinson’ had the highest proportion of water separation. Though we lacked a quantitative method to measure texture, the consistency of processed ‘Fipushi’ resembled ‘Dickinson’ the most (Figure 4). Comparatively, the majority of C. maxima fruit evaluated were significantly drier than ‘Dickinson’.
Table 1. Results of winter squash (Cucurbita moschata) breeding line field evaluations. Yield parameters were calculated per ten plants. Statistically significant differences between accessions were determined with the Tukey’s HSD test, at P=0.05. Numbers followed by the same letter are not significantly different.
Table 2. Results of field evaluations of a winter squash (Cucurbita maxima) diversity panel and commercial controls. Yield parameters were calculated per ten plants. Statistically significant differences between accessions were determined with the Tukey’s HSD test, at P=0.05. Numbers followed by the same letter are not significantly different.
Table 3. Results of C. moschata breeding line processing evaluations. Statistically significant differences between accessions were determined with the Tukey’s HSD test, at P=0.05. Numbers followed by the same letter are not significantly different.
Table 4. Results of C. maxima breeding line processing evaluations. Statistically significant differences between accessions were determined with the Tukey’s HSD test, at P=0.05. Numbers followed by the same letter are not significantly different.
Figure 1. Representative fruit from the powdery mildew resistant parent ‘Bugle’, the susceptible parent ‘Dickinson’, and the progeny with promising disease resistance and canning qualities. Photos: Elizabeth Indermaur, Cornell AgriTech
Figure 2. Representative fruit from Cucurbita maxima breeding lines with promising powdery mildew
tolerance. Photos: Elizabeth Indermaur, Cornell AgriTech
Figure 3. Jars from ‘Dickinson’, ‘Bugle’, and Accession 3 with Libby’s Canned Pumpkin for comparison. Photos: Elizabeth Indermaur, Cornell AgriTech
Figure 4. Jars from ‘Dickinson’, ‘Kestane’, ‘Fipushi’, and ‘Autumn Cup F1’ with Libby’s Canned Pumpkin for comparison. Photos: Elizabeth Indermaur, Cornell AgriTech
Honeynut is a miniature butternut noted for its quality, but because it is late maturing, it’s quality can be impacted by powdery mildew that infects the plant during carbohydrate filling. Previously, crosses with a source of Pm-0 for powdery mildew resistance were selected for several generations for quality and improved storage. This resulted in multiple new cultivars, however they lack the characteristic dark rind color of ‘Honeynut’ that functions as both a control for quality and consumer recognition. We used the Pm-0 marker to select for homozygous resistant individuals within BC2F2 populations, assayed them for storage, aesthetics and eating quality and will select within these homozygous PMR breeding lines going forward.
Introgress, pyramid/stack resistances into advanced breeding lines | Phytophthora
To identify segregating interspecific squash populations which had captured Phytophthora crown and root rot resistance from ‘Dickinson’, a seedling stage, greenhouse trial at Michigan State University Plant Science Greenhouses in East Lansing, MI was established. The trial consisted of a total of 8 interspecific breeding populations (BC2F1) and 3 parental lines provided by Dr. Mazourek (Cornell Univ.). On 26-May, 18 seeds per line were directly seeded onto 3x3in plastic containers containing SureMix soilless medium. On 29-June, when seedlings were at 3-4 leaf stages, all seedlings were inoculated with a zoospore suspension (2×104/ml) of Phytophthora capsici (M. Hausbeck P. capsici collection, strains SP98 and 12889). Lines were distributed in a complete randomized block design with three replications. Disease ratings based on a 0-5 categorical scale (0= healthy, 1=small lesion at crown, 2= water-soaked lesions beyond cotyledons, 3=wilt and plant partially collapsing, 4= severe wilt and pant completely collapsing, and 5=plant death) were conducted once a week after inoculation on 7-, 13-, and 20-July.
To evaluate squash cultivars and breeding lines for Phytophthora crown and rot resistance under Michigan’s field conditions, a total of 30 squash breeding lines received from Cornell University (Mazourek: 16 entries) and University of Florida (Meru: 14 entries) were included in two independent trials (Trial 1 and Trial 2). Field plots were established at SWMREC, Benton Harbor, MI. Prior to planting, fields were prepared following commercial production standards: the sandy soil was plowed and disced, and amended with nitrogen (100lb/A), potassium (180lb/A), sulfur (25lb/A), and boron (2lb/A) on 31-May. For Trial 1 (Mazourek), on 7-Jul., thirty 3-week-old seedlings per line were transplanted onto raised beds covered with black polyethylene plastic at 18in apart from each. For Trial 2 (Meru), on 16-Aug., twenty 3-week-old seedlings per line were transplanted onto raised beds at 18in apart from each. Lines for each trial were distributed in a complete randomized block design with four (Mazurek) or three (Meru) replications. On 22-Jul. and 30-Aug. for Trial 1 and Trial 2, respectively, each plant was inoculated with 1 g of P. capsici (M. Hausbeck P. capsici collection, strains SP98 and 12889) at a 1:1 ratio) infested millet placed at the crown of each plant. On 12-Aug. and 12-Sept., the first symptoms of Phytophthora crown rot were observed in the susceptible control lines; 22-T1B-13 (‘Golden Delicious’) for Trial 1 and ‘Early Prolific’ for Trial 2. Plant death assessments were performed on; 12-, 19-, 26-Aug. and 1-, 6-, 13-Sept. for Trial 1, and 7-, 20-, and 27-Sept. for Trial 2. For both trials, the area under the disease progress curve (AUDPC) was calculated at the end of the season and data were analyzed with SAS statistical software, version 9.4, using the PROC GLIMMIX procedure for a one-way ANOVA, with mean separation performed using Fisher’s least significant difference (LSD) with a Tukey’s adjustment.
For Trial 1, on 12-August plant death in the susceptible line 22-T1B-13 (‘Golden Delicious’) was 1.9% and increased significantly on 13-September (75.3%). On 13-September, 5 lines (22-
T1B-08, 22-T1B-06, 22-T1B-09, 22-T1B-10, and 22-T1B-11) were as susceptible as ‘Golden Delicious’. Five lines (22-T1B-02, 22-T1B-04, 22-T1B-05, 22-T1B-07, and 22-T1B-12) were as resistant as the intermediate F1 lines 22-T1B-014 and 22-T1B-15 (‘Autumn Cup’ and ‘Thunder’, respectively) and the resistant line 22-T1B-16 (‘Dickinson’). Resistance on 2 out of the 12 experimental lines tested (22-T1B-01 and 22-T1B-03) is still under segregation since plant death was not different than susceptible and resistant control lines. According to the area under the disease progress curve (AUDPC), 4 out of the experimental lines evaluated (22-T1B-02 22-T1B04, 22-T1B-05, and 22-T1B-07) show promising levels of resistance to P. capsici.
For Trial 2, on 12-September plant death in the susceptible cultivar ‘Early Prolific’ was 44.4% and increased rapidly and significantly on 20-September (94.4%). On 27-September, one line (358-174) shown moderate resistance as moderate resistant controls (PI181761-36p-Lot 1 and PI181761-36p-Lot 3) and resistant control (SSS333-7). Three lines (SS2147, SS2071, and SS2078) were as resistant as the resistant controls (SS333-8 and SS69-72). According to the area under the disease progress curve (AUDPC), 3 out of the 10 experimental lines evaluated (SS2147, SS2071, and SS2078) show promising levels of resistance to P. capsici and 3 lines (358-195, SS792-2, and 358-164) are still under segregation.
To evaluate breeding lines for Phytophthora fruit rot Age-Related Resistance (ARR), fruits from a total of 16 squash cultivars were collected from the field and infected with P. capsici (strain SP98) under laboratory conditions. A pollination plot was established at MSU-PPF, East Lansing, MI and prepared following commercial production standards; the Capac loam soil was plowed and disced on 20-May and 1-Jun., respectively, and amended with 100 lb Urea and 45 lb Potash on 1-June. On 7-Jul., transplants (30 seeds per cultivar or line) were directly seeded every 18 in onto raised beds (rows 100ft X 16ft center to center) covered with black polyethylene plastic. Until fruits were harvested, 28% fertilizer (1gal/A) was applied weekly and non-target diseases and insects were controlled. Starting on 7-Sept., a mixture of Torino (3.4 fl oz) and Bravo (32 fl oz) was applied, as needed, to control the incidence of powdery mildew, and Admire Pro (10.5 fl oz) was delivered through drip tape to control insects. To facilitate fruit set and reduce natural flower abortion, flowers at anthesis were hand-pollinated using an artist’s paint brush and marked at the petiole using colored-flagging tape. Because flower development among the lines was asynchronized, the presence of flowers at anthesis in each line was monitored daily for 30 days and hand-pollination was performed every other day during this time. A total of 790 flowers were hand-pollinated to harvest a maximum of 12 fruits
corresponding to 21 days post-pollination (dpp) and 12 fruits corresponding to 28dpp per line. A 4 mm mycelial plug of a 7-day-old culture of P. capsisci (strain SP98) was placed onto the surface/epidermis of each disinfected fruit on same day each fruit was harvested. Fruit rot was evaluated 5 days post-inoculation (dpi) and disease assessment included: 1) lesion size, 2) incidence of hyphae beyond inoculation point, and 3) disease severity based on a 0-4 categorical scale (0= healthy, 1=water-soaked lesion, 2= light mycelial growth, 3=moderate mycelial growth, 4= severe mycelial growth).
Overall, fruits corresponding to 3 breeding lines (22-T1A-02, 22-T1A-11, and 22-T1A-12) show a reduction of disease incidence (%) over time. A 30-60% incidence of fruit rot was observed when fruits corresponding to 21dpp were inoculated with P. capsici, however fruit rot was not observed for fruits corresponding to 28dpp from the 3 experimental lines indicated above. These results indicate age-related resistant for these 3 lines as previously observed for resistant control lines 22-T1A-14 (‘Autumn Cup’) and 22-T1A-16 (‘Dickinson’).
The main causal agent of powdery mildew in C. moschata in Puerto Rico was identified as Podosphaera xanthii. through a combination of molecular and morphological approaches, amplification of the ITS 5.8 rDNA region and conidia with had elliptical shape, presence of fibrosin bodies and either forked or apically swollen germination tubes.
Resistance to powdery mildew (Posphaera xanthii) was evaluated in Cornell Pm-0 derived resistant lines and local tropical pumpkin (Cucurbita moschata) genotypes. These included ‘Taína Dorada’, ‘Soler’, ‘Verde Luz’, ‘Waltham’, ‘Dickinson’, 20-1716-05 x 1720-05, 20-1716-02 x 1720, 20-171608 x 1720-02 and 20-1716-03 x 1720-02. The field trial was a replicated complete bock design with 3 reps and inoculation was perfomed with infected leaf suash applied to the top of young leaves. Disease severity and incidence were recorded in addition to agronomic traits. Vigor and vine length was greater in tropical pumpkins. ‘Taína Dorada’ and ‘Soler’ had significantly more fruit (p<0.05) ‘TD’ had significantly more yield (p>0.05). The Incidence of PM was highest (50%) in ‘Taína Dorada’ and ‘Waltham’ (p<0.05) while the severity of PM was highest in ‘Waltham’, 20-1716-02×1720 and Dickinson (p<0.05). In a greenhouse trial, the incidence of PM was highest (80-89%) in temperate genotypes (p>0.05) and severity was highest in ‘Ponca’ and all temperate genotypes (p>0.05).
A breeding program was initiated at UPR for PMR tropical C. moschata. Parental genotypes were assayed for resistance and intermated to create different breeding populations. Greenhouse trial results indicate that under high pathogen pressure Ponca and all Cornell genotypes had significantly higher PM infection than local genotypes. Local genotypes, seem to be more tolerant to PM (P. xanthii).
