Developing a Greenhouse Protocol for Evaluating Resistance to Corynespora cassiicola in Cotton (Gossypium hirsutum)

Target spot, caused by Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei, has reemerged as a problematic disease on cotton, Gossypium hirsutum L., throughout the Southeast and Mid-South United States (Mehl et al. 2020; Sumabat et al. 2018). The disease was first described on cotton in Mississippi in 1961; however, published reports of target spot were relatively uncommon until 2005 (Fulmer et al. 2012; Jones 1961). Necrotic, irregular foliar lesions with concentric rings are characteristic of target spot, and lesions are initially observed in the lower to middle canopy. Target spot is usually first noticed in cotton fields when the canopy closes between planted rows (i.e., when foliage covers bare soil between rows) and has been observed as early as 60 days after planting. Lesion numbers and size increase over time, particularly in warm, wet, or humid conditions, and affected leaves in the lower to middle canopy prematurely defoliate (Mehl et al. 2020). Defoliation can be as high as 70 to 80% and has been observed to reduce yield by several hundred kilograms of lint per hectare (Bowen et al. 2018; Fulmer et al. 2012).

The current management strategy for target spot of cotton is fungicide application; greater yield has been observed on some cultivars with a single fungicide application (Bowen et al. 2018; Hagan et al. 2018; Mehl et al. 2020). It appears that appropriate timing for a fungicide application is within 14 days of target spot onset, but it can vary depending on canopy density, the weather, and other factors not yet identified (Mehl et al. 2020). A meta-analysis of data from research conducted at 15 site-years indicated that a yield gain of 4 to 6% can be realized with such a timely fungicide application (Mehl et al. 2020). However, most states annually estimate <0.5% cotton yield losses as a result of C. cassiicola and other foliar fungal pathogens (National Cotton Council 2020). With cotton production valued at >$6 billion in the United States (U.S. Department of Agriculture National Agricultural Statistics Service 2020b), unrealized returns when target spot is not appropriately managed could amount to estimated losses of >$15 million.

Relying solely on fungicides for managing target spot of cotton is imprudent because fungicide resistance has been observed in C. cassiicola on cucumber (Miyamoto et al. 2009) and tomato (Vallad 2011). In addition, inappropriate application timing may cost more than is realized with greater yield (Mehl et al. 2020). Partial or full resistance of a cotton cultivar to C. cassiicola is the most efficient way to manage defoliation attributable to target spot (Hagan 2014; Hagan et al. 2018). In 2013, a study in southwestern Alabama determined that cotton cultivars vary in genetic resistance. An apparently susceptible cultivar, PhytoGen brand PHY 499 WRF (PHY 499; PhytoGen Cottonseed; Dow AgroSciences, Indianapolis, IN), had an average 448 kg of lint/ha loss (50% of average yield in the United States; U.S. Department of Agriculture National Agricultural Statistics Service 2020a), whereas a less susceptible cultivar, Deltapine brand DP 1252 B2RF (Deltapine Cottonseed; Bayer, St. Louis, MO), had losses of 269 kg of lint/ha (Hagan et al. 2015) compared with nontreated controls. However, based on timing of disease onset or the degree to which disease develops, yield losses can vary; for example, Bowen et al. (2018) noted that PHY 499 had 13 to 30% yield losses over 4 site-years. The cotton cultivar PHY 499 had been planted to nearly 30% of southeastern United States hectares in 2012 (U.S. Department of Agriculture Agricultural Marketing Service 2012), likely because of its consistently greater yields than other cotton cultivars in the absence of target spot or with an appropriate fungicide program (Hagan et al. 2018; Mehl et al. 2020).

In any year, several cotton cultivars dominate the market, and cultivar availability changes annually (U.S. Department of Agriculture Agricultural Marketing Service 2015). New cultivars that are introduced may have higher yield potential, new technologies (e.g., Bt) or resistance traits (for herbicides or diseases), or other favorable characteristics. For example, resistance to bacterial blight (caused by Xanthomonas axonopodis pv. malvacearum) is desirable in a cotton variety, and cotton lines and variety trials are regularly screened for this trait (Bourland 2018). Foliar fungal diseases, however, have not been problematic on cotton in the recent past, and selection for resistance to such diseases has not been a focus of cotton breeding programs.

To identify sources of pathogen resistance in a breeding program, numerous lines need to be evaluated, and in field settings, disease may not occur consistently. Thus, there is a need to develop a method to evaluate cotton genotypes in a controlled environment (e.g., a greenhouse), and it would be highly desirable to work with cotton seedlings rather than adult plants for rapid evaluation. To develop such a protocol, knowledge of favorable temperatures and wetness duration required for C. cassiicola infection of cotton is critical.

C. cassiicola has a broad host range and is a common pathogen in the tropics and subtropics (Dixon et al. 2009). Previous work has reported that C. cassiicola conidial germination is favored at relatively high temperatures (25 to 30°C) regardless of the host origin (Ahmed et al. 2013; Fernando et al. 2012; Seaman et al. 1965). However, some disparity exists relative to the duration of leaf wetness needed for C. cassiicola conidial germination and infection. For example, as little as 6 h of leaf wetness allowed disease development by C. cassiicola on Lantana camara (Pereira et al. 2003), but on tomato (Solanum lycopersicum L.), >16 h of leaf wetness was needed for disease development (Jones and Jones 1984); these differences may be attributable to the different hosts. Given that isolates of C. cassiicola from cotton are reported to be genetically distinct from those of other hosts (Sumabat et al. 2018), cotton isolates may have a different set of favorable environmental conditions for infection and disease development than isolates from other hosts.

Therefore, the objectives of this study were to determine optimal temperatures and leaf wetness periods for target spot occurrence and develop a protocol to evaluate numerous cotton genotypes.

Materials and Methods

C. cassiicola isolation and inoculum preparation.

Cotton leaves with lesions symptomatic of target spot were collected in late August 2015 and early September 2017 from the Prattville Agricultural Research Unit (32.427, −86.447) (isolate CC1) and the Brewton Agricultural Research Unit (31.142, −87.050) (isolate BRW1), respectively. A diseased leaf was maintained in a moist chamber for 24 h, after which conidia were removed from a lesion using a sterile needle and spread on water agar, which allowed immediate selection of individual conidia. Single spores were moved to V8 juice agar (200 ml of clarified V8 juice, 2 g of calcium carbonate, and 15 g of agar/liter of water; Jeffers 2006) and maintained at 28°C.

Colonies of C. cassiicola for conidia production were produced on quarter-strength potato dextrose agar (qPDA; 9.75 g of PDA powder and 11.25 g of agar/liter). Plugs (5 mm in diameter) from 12-day-old cultures were placed on fresh qPDA and maintained at 28°C; conidia developed in approximately 12 days. A conidial suspension was prepared by flooding the culture dish with a sterile 0.01% Tween 20 solution (Thermo Fisher Scientific, Waltham, MA) and lightly scraping mycelia with a sterile L-shaped glass rod to detach conidia. The resultant spore suspension was filtered through four layers of cheesecloth. Tan-colored, elongated conidia (100 to 180 µm long) with a slight taper in width lengthwise were counted using a hemocytometer and adjusted to desired concentrations with a sterile solution of 0.05% Tween 20.

Molecular confirmation.

To confirm that the established isolates were C. cassiicola, nucleic acid sequences were compared with NCBI records. Morphological characteristics of the two isolates were similar to one another, and BRW1 was arbitrarily selected for sequencing. Briefly, the isolate was grown on qPDA and incubated at 28°C until the plates were covered with fungal growth (11 to 15 days). Mycelia were scraped off the plate using a scalpel, a 50-mg sample was placed in a 2-ml tube with five 2.8-mm ceramic beads (Bead Ruptor Elite; Omni International, Kennesaw, GA), and the sample was homogenized for 2 min. DNA extraction was performed using an E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-tek, Norcross, GA) according to the manufacturer’s protocol except for the use of fresh tissue rather than dried. PCR was conducted using the extracted DNA as a template and primer pair ITS1 and ITS4 (Raja et al. 2017). A band size of approximately 550 bp was cut from the gel and purified using the E.Z.N.A. Gel Extraction Kit (Omega Bio-tek). The sequence was compared with NCBI GenBank entries using the BLAST search.

Effect of temperature and leaf wetness duration on target spot of cotton development.

Seed of the upland cotton cultivar PHY 499 was placed in a moist paper towel, and after 2 days, germinated seed was planted in potting mixture (Sun Gro Professional Growing Mix; Sun Gro Horticulture, Agawam, MA) in 9.5 × 9.5 cm² plastic pots. Plants were maintained at 28°C in a growth chamber with fluorescent lighting on a diurnal cycle of 14 h on/10 h off. Plants were watered as needed by drenching the soil surface. When plants were approximately 24 days old with three to four true leaves, each was inoculated with CC1. Using a pipette, five drops of a conidial suspension (4 × 10⁴ spores/ml) were placed on each of three true leaves of a plant. Inoculated leaves were covered with wet tissue (Kimwipes; Kimberly-Clark, Roswell, GA), and the entire plant was enclosed in a plastic bag.

To determine optimal temperatures for C. cassiicola infection of cotton, 21 bagged plants were placed in each growth chamber, each chamber with a different temperature. Five temperatures were evaluated: 16, 20, 24, 28, and 32°C. At 8, 12, 16, 24, 32, 40, and 48 h postinoculation, bags and wet tissue were removed from three replicate plants at each temperature. Uncovered plants were placed back into the temperature from which they were removed. Plants were examined at 24-h intervals for 9 days after inoculation (DAI), and day of disease onset was recorded (as DAI). At 6 DAI, numbers of lesions were counted on the three inoculated leaves of each plant. The experiment was performed twice.

Greenhouse protocol development.

Four genotypes were selected from the 2017 Regional Breeders Trial Network (RBTN) in Fairhope, Alabama. Two were classified as resistant (NM 16-13P1088B and LA 14063001) and two as susceptible (PHY 499 and TAM LBB130218) based on symptom ratings in the field (Koebernick 2017). A conventional cultivar, UA 48, which demonstrated at least tolerance to C. cassiicola in the 2016 growing season (J. Koebernick, personal observation), was included. Seed of each genotype was planted into 11.5 × 11.5 cm² square pots using the same medium as used in growth chamber trials. Three seeds were sown in each pot; after emergence, seedlings were thinned to one per pot. Four replicate pots of each genotype plus four additional pots of noninoculated PHY 499 were arranged in a randomized complete block on a greenhouse bench. The noninoculated PHY 499 pots were used to ensure that what was rated as target spot was a result of inoculation. A soil drench of imidacloprid (Admire Pro, Bayer Crop Science, Research Triangle Park, NC) was applied at 20 μl per plant to reduce insect pressure. In the greenhouse, 12 h (06:00 to 18:00) of supplemental lighting was provided by 1,000-watt metal halide lamps. Average daily temperatures during these evaluations were 20 to 28°C.

When cotton cotyledons had fully emerged from the soil (10 to 14 days), plants were inoculated by spraying approximately 1.5 ml of BRW1 conidial suspension (4 × 10⁴ conidia/ml just to run-off) onto cotyledons; the suspension was prepared as previously described. The isolate BRW1 was used for these trials because of contamination problems with CC1. Inoculated plants dried for no more than 5 min and were subsequently placed in a mist chamber with the noninoculated PHY 499. The mist chamber was built of PVC (2.5 × 1.2 × 1.2 m) and enclosed in plastic sheeting with three misting nozzles near the ceiling of the chamber (Beckman and Payne 1983). Mist operated for 1 s every 10 min, allowing leaves to stay consistently wet without run-off. Because of nonoptimal, fluctuating temperatures in the greenhouse, the mist system operated for 72 h. At 14 DAI, disease severity across the two cotyledons on each plant was estimated with the help of severity diagrams (James 1971) and recorded. The second and third trials, with the same five genotypes previously evaluated, included eight replications of each genotype.

Data analysis.

Generalized linear mixed model (GLMM) analysis (PROC GLIMMIX, SAS v. 9.4; SAS Institute Inc., Cary, NC) was used to examine the effect of temperature, moisture duration, and interaction of temperature and moisture on time of disease onset and number of lesions. Each replicate and replicate-temperature combination was considered a random effect in the models. For greenhouse data, a GLMM was specified to determine differences in disease severity among genotypes, and replicate was considered a random effect in these models. The last two greenhouse trials were identical to one another and were combined for analysis. Again, a GLMM was used to examine the effect of genotype and trial on disease severity, with replication and replication-trial combinations as random effects in the model. All GLMMs were fitted with a normal response distribution and identity link function. Factor effects were considered significant when P ≤ 0.05; means separations were based on Fisher’s protected least significant difference test.

Results

Molecular confirmation.

The DNA fragment sequence selected by IST1 and IST4 primer pairs revealed a 99% match to known C. cassiicola sequences in NCBI (accession nos. AY238606 and JQ717069). Our results confirmed beyond visual identification that the BRW1 isolate is C. cassiicola.

Effect of temperature and leaf wetness duration on target spot of cotton development.

Disease onset was significantly affected by temperature (P < 0.0001), leaf wetness duration (P < 0.0001), and their two-way interaction (P < 0.0001). Disease onset was earliest with 28°C and >16 h of wetness (Table 1). Target spot was not observed over the 9-day evaluation period with <16 h of wetness except at 28°C. Onset was delayed to ≥3 DAI with <40 h of wetness duration at 16°C, 16 h of leaf wetness at 20 and 24°C, <16 h of wetness at 28°C, and for all leaf wetness periods at 32°C (Table 1).

Table 1. Days after inoculation for target spot onset on cotton in growth chamber studies with different temperatures and leaf wetness durations

Moisture duration (h)	Temperature (°C)
	16	20	24	28	32
8	–y	–	–	5.7 a	–
12	–	–	–	3.5 c	–
16	–	3.6 bc	3.1 c	2.0 cd	–
24	5.2 abz	2.9 cd	1.9 cd	1.0 d	–
32	3.0 c	1.9 cd	1.9 cd	1.0 d	3.5 c
40	2.5 cd	1.9 cd	1.9 cd	1.0 d	3.7 bc
48	2.0 cd	1.9 cd	1.9 cd	1.0 d	3.5 c

^y Inoculated plants were evaluated for 9 days. A dash indicates that disease was not observed during the 9-day trial.

^z Letters following means, when different, indicate significant differences according to Fisher’s protected least significant difference at P ≤ 0.05.

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The number of lesions that developed by 6 DAI differed significantly (P < 0.0001) because of temperature, wetness duration, and the temperature × wetness interaction. For each moisture period, the number of lesions that were observed at 6 DAI was consistently greatest when plants were maintained at 28°C (Fig. 1); the lowest numbers of lesions developed at the coolest (16°C) and warmest (32°C) temperatures evaluated, whereas intermediate numbers of lesions were noted at 20 and 24°C. Longer periods of wetness generally allowed greater lesion numbers (Fig. 1).

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Greenhouse protocol development.

Genotypes differed significantly (P = 0.0002) in their disease reactions in the first greenhouse trial (Fig. 2). The inoculated PHY 499 had the greatest severity (93.8%), whereas the noninoculated PHY 499 had no disease (Fig. 2). Disease severity on TAM LBB130218 was similar to that on PHY 499. The cultivar UA 48 had target spot severity that was similar to that on cultivars LA14063001 and NM 16-13P1088B (Fig. 2).

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Reductions in target spot severity were noted in the second and third trials across all genotypes. However, these two trials did not differ significantly (P > 0.21) from one another although they were conducted at different times in the greenhouse. Target spot differed significantly (P < 0.0001) among genotypes, and the trial × genotype interaction was not significant (P = 0.3572). Target spot on PHY 499 was significantly greater than on all other genotypes (Fig. 3). Less disease was seen on LA14063001 compared with PHY 499 and TAM LBB130218. Remaining genotypes had intermediate disease levels (Fig. 3).

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Discussion

The current study evaluated the effect of temperature and wetness duration on the onset and lesion development of target spot of cotton. Lesions resulting from infection by C. cassiicola were noted at 1 DAI at 28°C, but disease developed, albeit more slowly, at all temperatures evaluated (16 to 32°C). Our results indicate that the optimal temperature for target spot development is approximately 28°C, which concurs with the favorable temperature range of 25 to 30°C reported for conidial germination of isolates of this fungus from okra [Abelmoschus esculentus (L.) Moench] (Ahmed et al. 2013), which is a cotton relative, and tomato (Jones and Jones 1984).

We observed disease onset at 1 DAI on CC1-inoculated plants when maintained at 28°C with ≥24 h of leaf wetness. Fulmer et al. (2012) maintained inoculated cotton seedlings for 48 h in a moist chamber at 21.1°C and noted disease onset within 1 week of inoculation. Similarly, Conner et al. (2013) observed disease onset at day 6 when inoculated plants were maintained at 21°C in a moist chamber in the greenhouse for 72 h. Temperature differences among these reported research studies could explain the difference in timing of apparent onset of target spot on cotton.

The greatest lesion counts on cotton were observed at 28°C with 48 h of leaf wetness (maximum leaf wetness duration tested). Seaman et al. (1965) reported that day and night temperatures of 25 ± 2°C and 22 ± 2°C, respectively, with 48 h of moisture resulted in numerous pinpoint lesions on soybean [Glycine max (L.)] foliage. Similarly, Jones and Jones (1984) reported that 20 to 28°C was a favorable temperature range for target spot on tomato, with the greatest disease severity observed at 24 and 28°C. Our results indicate that the favorable temperature for target spot of cotton coincides with those observed for target spot of soybean and tomato.

Our studies determined that shorter periods of leaf wetness (i.e., 8 and 12 h) were sufficient for disease onset at optimal temperatures (28 and 24°C, respectively). However, at temperature extremes of 16 and 32°C, target spot only developed with extended periods of leaf wetness. In preliminary trials, moistened cotton leaves dried rapidly in growth chambers, even when plants were placed in sealed boxes with standing water. To overcome this drying, inoculated leaves were covered with wet Kimwipes to maintain leaf wetness. Germinating conidia, particularly with shorter periods of leaf wetness, may not have been established in plant tissue and could have been detached when the paper tissue was removed from leaves. Thus, it may be that lesion counts from growth chamber studies are reduced more than might be observed in a field setting. In reality, the microclimate within a cotton canopy is variable yet likely to be substantially more conducive for target spot development than the controlled conditions of growth chambers. Nevertheless, results of these studies indicated the need for relatively long leaf wetness duration, given that greenhouse temperatures fluctuated between 18 and 26°C; this was achieved with the mist system.

For a preliminary trial, we were unable to obtain >1.5 × 10⁴ conidia/ml, which did not allow separation of genotypes in the greenhouse. Because of time constraints, we did not repeat the use of this conidial density and used C. cassiicola inoculum at 4 × 10⁴ conidia/ml moving forward, as done in growth chamber trials. A lower inoculum density could have skewed genotype separation, or this lack of separation with the reduced inoculum density could have been an anomaly. Additional research in the future could evaluate different inoculum densities relative to genotype response to C. cassiicola infection.

In the first greenhouse trial with four replications, PHY 499 had disease similar to that of TAM LBB130218, and these two genotypes had the greatest target spot severities of evaluated lines, similar to what was seen in the 2017 RBTN (Koebernick 2017). However, variation in severity on cotyledons was high, ranging from 50 to 100% on TAM LBB130218 and 25 to 75% on NM 16-13P1088B. To gain precision, eight replications were used in two additional trials. In these last trials, PHY 499 had greater disease than TAM LBB130218, which had significantly greater disease than LA14063001; these results reflected genotype rankings seen in the 2017 RBTN (Koebernick 2017). The substantially reduced levels of disease on TAM LBB130218 as compared with PHY 499 in these trials differed from what was observed in the field, but the relative rankings (more susceptible to less susceptible) were similar for greenhouse trials and the 2017 RBTN. The similarity of rankings between field disease scores and disease on cotyledons in the greenhouse is an important consideration because resistance at the cotyledon stage may be distinct from resistance in adult plants (Ellis et al. 2014).

The cultivar PHY 499 has previously been reported to be more susceptible to C. cassiicola than other cultivars (Hagan et al. 2018; Mehl et al. 2020), but little is known about the susceptibility of the other genotypes contained in the RBTN. The current project focused on the ability to separate genotypic reactions to target spot in a controlled environment. Our intent was not to characterize specific breeding lines for their reaction to target spot because breeding lines in public programs are typically only tested for a single year as a result of their overall agronomic performance. Within a breeding program, large numbers of lines are evaluated against each other and, in general, only the top performing ones are reevaluated. Lines with a response to target spot on par with PHY 499 would be deemed undesirable. If a line shows promise relative to yield and/or resistance to C. cassiicola, it may be repeated in the next year’s RBTN and may require additional testing for genotypic response to target spot.

In a breeding program, numerous genotypes need to be evaluated for specific characteristics such as pathogen resistance. We evaluated a rapid screening protocol for assessing relative resistance of cotton to C. cassiicola using cotton seedlings under mist irrigation. Fully expanded cotyledons were noted by day 14 after planting, and disease reactions of inoculated cotyledons were evaluated 14 days later; thus, each trial for evaluating genotypic differences took approximately 1 month. The relative susceptibility rankings of genotypes with this system agreed with that noted in the 2017 RBTN (Koebernick 2017). The protocol, as outlined herein, will allow rapid processing of numerous genotypes and may be useful for screening for resistance to other fungal foliar pathogens in other crops.