Phytophthora capsici annually threatens production of cucurbit and solanaceous crops. Long-lived oospores produced by the pathogen incite primary infection of susceptible plants when conditions are wet. Limiting the rot of winter squash and pumpkin (Cucurbita sp.) fruits is difficult due to the long maturation period when fruits are often in direct contact with infested soil. Genetic resistance to fruit rot is not widely available within Cucurbita sp.; however, age-related resistance (ARR) to P. capsici fruit rot develops in specific cultivars during maturation. The objective of this study was to evaluate the fruits of 12 cultivars of Cucurbita pepo, Cucurbita moschata, and Cucurbita maxima for ARR to P. capsici using a mycelial-plug inoculation method. All Cucurbita pepo and Cucurbita moschata cultivars displayed ARR; 7 days postpollination (dpp) fruits were susceptible, limited lesion development occurred on fruits 22 dpp, and lesions did not develop at 56 dpp. Disease developed on both Cucurbita maxima cultivars tested at 7, 14, 22, and 56 dpp. Firmness of fruit exocarps was measured with a manual penetrometer. Exocarp firmness of all cultivars increased during maturation; however, there was no correlation between firmness and disease incidence among cultivars at 22 dpp (R2 = −0.01, P = 0.85). When fruits of cultivars expressing ARR at 22 dpp were wounded before inoculation, fruit rot developed.
Phytophthora capsici is a destructive pathogen of cucurbit and solanaceous vegetables. All cultivars of squash are considered susceptible to phytophthora root, crown, and fruit rot (Babadoost and Islam, 2003; Cafe et al., 1995); losses in winter squash and pumpkin (Cucurbita sp.) production have exceeded 50% (Babadoost, 2000; Isakeit, 2007; Meyer and Hausbeck, 2013a). The pathogen overwinters in the soil and plant residue as long-lived oospores that serve as primary inoculum. The polycyclic production of sporangia and zoospores occurs on infected plant tissue. Movement of P. capsici in surface water used for irrigation has contributed to the dispersal of the pathogen in Michigan (Gevens et al., 2007). Managing phytophthora root and crown rot requires an integrated approach that includes raised plant beds in conjunction with fungicides applied via drip irrigation or soil-directed sprays (Foster and Hausbeck, 2010; Jones and McGovern, 1994; Meyer and Hausbeck, 2013b). Tolerance to root rot has been identified in cultivars of summer (Cucurbita pepo) and winter squash (Cucurbita moschata) and cucumber (Cucumis sativus) (Hausbeck and Lamour, 2004; Meyer and Hausbeck, 2012; Ppoyil, 2011). Raised bed culture with plastic mulch-covered plant beds can limit soil splash onto fruit (Kousik et al., 2011); however, vines of winter squash and pumpkin typically grow off of the plastic mulch coming into direct contact with the soil between the plants beds. Foliar fungicides to protect against fruit rot are limited by a dense foliar canopy and an inability to cover the fruit surface in contact with the soil (Newhall and Wilkinson, 1949). Additionally, raised plant beds covered with plastic mulch with trickle irrigation are not economical for growers of winter squash for processing where profit margins are narrow. Over 40,000 acres of winter squash and pumpkin grown in the Midwest (Anonymous, 2015) highlight the importance of developing effective strategies to limit fruit rot.
The ability of plants to acquire resistance to pathogens as they mature has been studied in many host–pathogen systems (Gadoury et al., 2003; Gerlach et al., 1976; Kennelly et al., 2005; Kim et al., 1989), especially resistance of seedlings to oomycete pathogens (Koh et al., 1987; Lazarovits et al., 1981; McClure and Robbins, 1942; Mellano et al., 1970). Vegetable crops in the Cucurbitaceae and Solanaceae families develop ARR to P. capsici fruit rot (Ando et al., 2009; Biles et al., 1993; Gevens et al., 2006; Meyer and Hausbeck, 2013a) and modifications to disease management programs have been suggested (Ando et al., 2009; Hausbeck and Lamour, 2004; Krasnow et al., 2014; Meyer and Hausbeck, 2013a). The fruits of cucurbit crops including acorn squash, pumpkin, and cucumber are highly susceptible to P. capsici during early fruit formation, but become increasingly resistant as they mature (Ando et al., 2009; Gevens et al., 2006). Watermelon, muskmelon, and summer squash do not appear to have appreciable levels of resistance (Ando et al., 2009). Meyer and Hausbeck (2013a) found differences in the onset and magnitude of ARR to P. capsici fruit rot between ‘Dickenson Field’ (Cucurbita moschata) and ‘Golden Delicious’ (Cucurbita maxima) processing squashes. Although both cultivars were susceptible to the pathogen up to 14 dpp, ‘Dickenson Field’ developed ARR at 21 dpp (<15% fruit rot), whereas ‘Golden Delicious’ remained susceptible (≈80% fruit rot). Mechanical harvesting of processing squash with incipient P. capsici infections can result in spread of the pathogen to surrounding fruit postharvest and during transportation, resulting in potential loss of entire truckloads (Hausbeck and Lamour, 2004; Kousik et al., 2014). The large acreage and low profit margin of squash grown for the processing market necessitate novel methods of disease control.
The differences in the onset of ARR among cucurbits (Ando et al., 2009; Gevens et al., 2006; Krasnow and Hausbeck, 2015) and the lack of ARR in cultivars of watermelon (Citrullus lanatus) and winter squash (Cucurbita maxima) (Kousik et al., 2012; Krasnow and Hausbeck, 2015; Meyer and Hausbeck, 2013a) have made it difficult to incorporate this feature into disease management programs. Fungicides are applied to pickling cucumbers during the period of rapid fruit growth when the fruit are highly susceptible to P. capsici (Hausbeck and Lamour, 2004). Identifying winter squash and pumpkin cultivars that express ARR could help growers make cultivar selections and time fungicide applications to protect developing fruit. The objectives of this study were to 1) evaluate winter squash and pumpkin cultivars (Cucurbita sp.) for ARR to P. capsici and 2) determine the effect of morphophysiological changes during winter squash and pumpkin fruit development on ARR. A brief report of this work has been published (Krasnow and Hausbeck, 2015).
Materials and Methods
Plant culture and fruit inoculation.
Twelve winter squash and pumpkin cultivars representing the three most economically important Cucurbita sp. were selected (Table 1). Seeds were planted into 72-cell flats containing soilless media (Suremix Michigan Grower Products Inc., Galesburg, MI) and grown for 3 weeks in a greenhouse with 27 °C day /25 °C night temperatures. Squash and pumpkin seedlings were transplanted into 15-cm raised plant beds covered with black polyethylene plastic at the Michigan State University Plant Pathology Farm in Lansing, MI. The soil type was a Capac loam that was previously cropped to pumpkin and had no history of P. capsici infestation. Watering was accomplished with trickle irrigation and plants were grown according to local commercial production standards for fertilizer and pest management (Bird et al., 2014). Once female flowers reached anthesis, male flowers were removed and used to pollinate female flowers of the same cultivar. Female flowers were tagged with the date of pollination and desired harvest age. Fruit were harvested 7, 14, 22, and 56 dpp, ages were selected based on developmental changes in fruit color, firmness, and size (Loy, 2004; Meyer and Hausbeck, 2013a). Following harvest, fruit were surface sterilized in 10% bleach for 5 min, rinsed with tap water, and air-dried on a laboratory bench. Fruit length from the apex to blossom end and the width at the fruit’s widest point were measured. Two P. capsici isolates obtained from the culture collection of M. Hausbeck at Michigan State University, East Lansing, MI, were used for fruit inoculation; OP97 (A1 mating type, sensitive to mefenoxam, isolated from pumpkin) and 12889 (A1 mating type, insensitive to mefenoxam, isolated from pepper). The isolates were grown on V8 juice agar (143 mL V8 juice, 3 g CaCO3, 16 g agar/L). To ensure isolate virulence, the isolates were used to inoculate squash fruit and subsequently recovered from the diseased fruit before the initiation of the study (Quesada-Ocampo and Hausbeck, 2010). To inoculate fruit, a 7-mm agar plug from the margin of an actively growing colony was placed mycelial side down in the middle of each fruit on the unwounded fruit surface. The agar plug was covered with a sterile plastic screw cap (Axygen Inc., Union City, CA) using petroleum jelly as a fixative to prevent plug desiccation. Control fruit were inoculated with sterile V8-agar plugs. Isolate OP97 was not used to inoculate 56 dpp fruit as there was not an adequate supply of the large-fruited Cucurbita sp. The inoculated fruit were incubated in large clear plastic bins (Sterilite, Townsend, MA) lined with moist paper towel to maintain high relative humidity (RH). WatchDog Dataloggers (Spectrum Technologies, Inc., Aurora, IL) were used to monitor temperature and RH within the bins. The average temperature and RH were 24.0 °C and 99.7%, respectively, during the study.
Cultivars, market use, and days to maturity of winter squash and pumpkin evaluated for age-related resistance to Phytophthora capsici fruit rot.
Fruit were removed from the bins 4 d postinoculation and lesion diameter was measured on two axes. Pathogen growth and sporulation were rated on a 0 to 4 scale adapted from Meyer and Hausbeck (2013a) where 0 = no visible pathogen growth; 1 = water-soaking only; 2 = light visible mycelial growth; 3 = moderate mycelial growth; and 4 = dense mycelial growth. Fruit receiving a mean rating value ≤0.5 were considered resistant (R), and fruit with a mean rating value >0.5 but <1.5 were considered intermediately resistant (IR). After disease assessment, 1- to 2-mm tissue sections were removed from the margin of diseased tissue and plated onto BARP (50 ppm benomyl, 100 ppm ampicillin, 30 ppm rifampicin, and 100 ppm pentachloronitrobenzene)-amended V8 agar plates. Recovered isolates were confirmed as P. capsici by pathogen morphology on V8-agar (Waterhouse, 1963). Mefenoxam sensitivity (Lamour and Hausbeck, 2000) was determined to verify similarity to the isolate used for inoculation. Control fruit were observed for symptoms and tissue samples cultured to confirm the absence of P. capsici infection. There were four fruit per replication per isolate with one control. The experiment was conducted twice.
Fruit firmness testing and wound assay.
Pericarp and exocarp firmness of healthy fruits were measured using a fruit penetrometer (model FT 327; QA Supplies LLC, Norfolk, VA) with a 5-mm-diameter press. The measurement was taken from squash or pumpkin tissue (≈25 cm2) after rating pathogen growth. Exocarp firmness was measured by using the fruit penetrometer to directly penetrate the exocarp. Pericarp tissue firmness was measured by removing the exocarp (0.5- to 1.0-mm depth) with a sterile scalpel before the measurement. For the wound assay, 22 dpp fruit from five cultivars representing each Cucurbita sp. were selected. Each fruit was wounded with a sterile needle to 1-cm depth before inoculation, and then incubated and assessed for disease as described previously. Isolate 12889 was used to inoculate fruit for the wound assay.
Data analysis was accomplished using SAS v9.3 (SAS Institute, Cary, NC). Differences among the variables including pathogen growth rating, lesion size, exo- and pericarp firmness, and fruit age were analyzed using analysis of variance in SAS Proc Mixed. Mean differences were separated using Fisher’s least significant difference (P = 0.05). Correlations among morphological features and disease incidence and severity at the four selected ages were analyzed with Pearson’s correlation coefficient (P = 0.05). Homogeneity of variance between isolates was assessed by residual analysis and data from each isolate were pooled as there were no significant differences in pathogen growth rating, lesion size, and disease incidence. Control fruit did not display symptoms after inoculation with sterile agar and were not included in the analyses.
The fruits of all winter squash and pumpkin cultivars tested increased in size and exo- and pericarp firmness as they matured from 7 to 56 dpp (Table 2). From 14 to 21 dpp, fruits increased in width for ‘Diablo’ (47%), ‘Hubba Hubba’ (25%), ‘Lumina’ (19%), and ‘Vegetable Spaghetti’ (14%); fruits from all other cultivars increased <5%. The length of fruits of all cultivars increased <15% from 14 to 21 dpp, with the exception of ‘Diablo’ (29%) (data not shown). At 22 dpp, ‘Hubba Hubba’ and ‘Lumina’ (Cucurbita maxima) had the least firm exocarp, whereas ‘Gold Dust’ and ‘Table Ace’ (Cucurbita pepo) had the firmest exocarp (Table 2). There was no correlation between exocarp firmness and disease incidence among cultivars at 22 dpp (r = −0.01; P = 0.85). Exo- and pericarp firmness was negatively correlated with disease incidence and pathogen growth when analyzed across all ages tested (r = −0.53; P < 0.0001). The exocarp of ‘Table Ace’ and ‘Gold Dust’ were the firmest among the cultivars tested at 14, 22, and 56 dpp (Table 2).
Exocarp firmness during development of select winter squash and pumpkin cultivars.
All cultivars were susceptible to P. capsici at 7 dpp with fruit rot incidence ranging from 69% to 100% (Table 3); pathogen growth was similar among cultivars (P = 0.241). ‘Vegetable Spaghetti’, ‘Avalon’, ‘Early’, and ‘Waltham’ were IR at 14 dpp with an average growth rating <1.5 (Table 3). ‘Autumn Delight’ and ‘Vegetable Spaghetti’ had the lowest disease incidence at this age (50%; Table 3). At 22 dpp, average disease ratings for all but two cultivars were <1 (Table 3) and disease incidence was >20% for six cultivars. Fruits from the two Cucurbita maxima cultivars Lumina and Hubba Hubba were the only ones to become infected at 56 dpp, with 63% and 25% fruit rot, respectively (data not shown).
Growth rating and disease incidence 4 d after inoculation with Phytophthora capsici of winter squash and pumpkin cultivars 7, 14, and 22 d postpollination.
Wounding the fruits of five cultivars before inoculation significantly increased disease incidence and pathogen growth (P ≤ 0.0001) compared with the unwounded inoculated fruits. ‘Vegetable Spaghetti’ had an average growth rating of <1 after wound inoculation due to a lack of pathogen sporulation and mycelial growth (Fig. 1); however, average lesion size was 4.1 cm, with 88% fruit rot incidence (Fig. 2). ‘Table Ace’, ‘Gold Dust’, ‘Waltham’, and ‘Hubba Hubba’ exhibited 100% fruit infection following inoculation of the wounded fruits, with an average rating of 3.0, 2.5, 3.5, and 3.6, respectively (Fig. 2). Superficial wounding of ‘Table Ace’ fruit by removing a thin piece of the exocarp <1.0 mm thick with a scalpel before inoculation resulted in 100% infection (data not shown).
The relatively long maturation time and growth habit of winter squash and pumpkin increases the risk of phytophthora fruit rot in growing regions with frequent rainfall and infested soil. Representative cultivar types of Cucurbita pepo, Cucurbita moschata, and Cucurbita maxima include jack-o-lantern pumpkin, butternut squash, spaghetti squash, and processing squash and all are susceptible to P. capsici fruit rot (Babadoost, 2000; Isakeit, 2007; McGrath, 2000; Meyer and Hausbeck, 2013a). Cultural practices including trellising and choosing varieties with a compact plant size can help prevent P. capsici fruit rot by avoiding contact with infested soil (Ando and Grumet, 2006). Large-fruited Cucurbita sp. offer unique challenges that are difficult to address using cultural practices. Exploiting ARR to P. capsici fruit rot offers a valuable opportunity to improve disease management when integrated with other strategies.
Most winter squash and pumpkin cultivars reach full size by 20 to 24 dpp (Loy, 2004) and the development of ARR in many of the Cucurbita sp. cultivars tested coincides with this period of growth (Krasnow and Hausbeck, 2015; Meyer and Hausbeck, 2013a). Similar to the results of experiments by Meyer and Hausbeck (2013a), the fruits of Cucurbita maxima cultivars in this study displayed a greater incidence of infection than the fruits of Cucurbita pepo and Cucurbita moschata cultivars. The Cucurbita maxima cultivars were the only Cucurbita sp. to develop P. capsici lesions at 56 dpp. Exocarp firmness of ‘Golden Delicious’ (Cucurbita maxima) and ‘Dickenson Field’ (Cucurbita moschata) processing squashes increased as the fruit matured, but firmness was not correlated with P. capsici lesion size on Golden Delicious, a cultivar highly susceptible to fruit rot (Meyer and Hausbeck, 2013a). In this study, there was no correlation between exocarp firmness and disease incidence among winter squash and pumpkin cultivars 22 dpp. Changes in surface wax as cucurbit fruit develop have been implicated as influencing resistance to P. capsici (Ando et al., 2009). Cucurbita maxima begins to accumulate epicuticular wax at 14 dpp (Sutherland and Hallett, 1993). Watermelon fruit also develop a thick wax layer at 14 dpp, which covers the fruit surface and stomat (Frankle and Hopkins, 1993). Fruits of Cucurbita maxima and Citrullus lanatus cultivars are susceptible to P. capsici at all maturity stages (Kousik et al., 2012; Krasnow and Hausbeck, 2015; Meyer and Hausbeck, 2013a), and changes in surface wax likely have a limited effect on ARR and fruit rot. Recent studies have identified Cucurbita pepo and Citrullus lanatus germplasm accessions with resistance to P. capsici fruit rot as early as 7 dpp during the period of fruit elongation (Kousik et al., 2012; Krasnow et al., 2014) providing additional evidence for the limited role of surface wax in resistance to fruit rot.
Biles et al. (1993) found that the cuticle of pepper increased in thickness as the fruit matured from green to red and developed resistance to P. capsici fruit rot. Similarly, the thicker cuticle and epidermal cells of the stem end of tomato were suggested to prevent infection of the fruit by P. capsici (Simonds and Kreutzer, 1944). The stylar end did not possess these characteristics and infection occurred within 70 to 90 min after inoculation. The exocarp of cucurbit fruit is likely the location where ARR is expressed as wounding the fruit before inoculation negates ARR. The surface of Cucurbita sp. contains trichomes and stomata (Barber, 1909; Sutherland and Hallett, 1993) and differences in the quantity and morphology of these structures may influence ARR to P. capsici. Zoospores were observed to accumulate preferentially over stomata of a Cucurbita maxima cultivar susceptible to P. capsici, but not a Cucurbita moschata cultivar with ARR (C. Krasnow and M. Hausbeck, unpublished data). Microcracks in the fruits surface also occur due to growth and water influx (Schaffer and Boyer, 1984) and may influence the susceptibility of winter squash and pumpkin cultivars to fruit rot.
Phytophthora fruit rot has long been a limiting factor in winter squash and pumpkin production. Cultivars that express ARR to P. capsici as early as 14 dpp could be selected as part of an integrated management program with fungicide sprays timed for the onset of fruit formation when protection is most needed. Winter squash and pumpkin may be stored postharvest before transporting and marketing. Infection and disease development of the fruit during postharvest storage are especially costly to growers due to the added expenses associated with disposing of the rotted produce. The use of cucurbit cultivars that express ARR may limit postharvest losses since ARR decreases the risk of fruit rot developing as the crop reaches maturity.
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