S. J. MacKenzie

Genetic and Pathogenic Analyses of Colletotrichum gloeosporioides Isolates from Strawberry and Noncultivated Hosts

ABSTRACT Colletotrichum crown rot of strawberry in Florida is caused primarily by Colletotrichum gloeosporioides. To determine potential inoculum sources, isolates of Colletotrichum spp. from strawberry and various noncultivated plants growing in the areas adjacent to strawberry fields were collected from different sites. Species-specific internal transcribed spacer primers for C. gloeosporioides and C. acutatum were used to identify isolates to species. Random amplified polymorphic DNA (RAPD) markers were used to determine genetic relationships among isolates recovered from noncultivated hosts and diseased strawberry plants. Selected isolates also were tested for pathogenicity on strawberry plants in the greenhouse. In all, 39 C. gloeosporioides and 3 C. acutatum isolates were recovered from diseased strawberry crowns, and 52 C. gloeosporioides and 1 C. acutatum isolate were recovered from noncultivated hosts. In crown inoculation tests, 18 of the 52 C. gloeosporioides isolates recovered from noncultivated hosts were pathogenic to strawberry. Phylogenetic analysis using RAPD marker data divided isolates of C. gloeosporioides from noncultivated hosts into two separate clusters. One cluster contained 50 of the 52 isolates and a second cluster contained 2 isolates that were homothallic in culture. Isolates from strawberry were interspersed within the cluster containing the 50 isolates that were recovered from noncultivated hosts. The results are not inconsistent with the hypothesis that C. gloeosporioides isolates obtained from strawberry and noncultivated hosts adjacent to strawberry fields are from the same population and that noncultivated hosts can serve as potential inoculum sources for Colletotrichum crown rot of strawberry.

Timing of Fungicide Applications for Botrytis cinerea Based on Development Stage of Strawberry Flowers and Fruit

During the 1999-2000 and 2000-2001 growing seasons, field experiments were conducted to identify the developmental stage(s) of strawberry flowers and fruit that requires fungicide applications to control Botrytis fruit rot. Fenhexamid, a protectant fungicide, was applied to individual newly opened flowers or fruit of cultivar Sweet Charlie at defined intervals after anthesis. In 1999-2000, a single application of fenhexamid at anthesis controlled Botrytis fruit rot as well as multiple weekly applications beginning at anthesis. During both seasons, disease control deteriorated as applications were delayed 7 and 14 days after anthesis. This trend was described by linear regression equations relating the time of application to Botrytis fruit rot incidence. Additional treatments tested the effects of emasculation and petal removal 3 to 7 days after anthesis. Emasculation significantly reduced disease incidence in 2000-2001. Petal removal produced modest but significant reductions in 1999-2000, but not in 2000-2001. These results demonstrate that strawberry flowers are more susceptible to Botrytis cinerea than green fruit, and suggest that stamens are the principal infection court. Fungicide applications should focus on peak bloom periods to minimize fungicide use and optimize control of preharvest Botrytis fruit rot. During these periods, applications should be made at close intervals (≤7 days) to minimize losses to Botrytis.

Postbloom Fruit Drop of Citrus and Key Lime Anthracnose Are Caused by Distinct Phylogenetic Lineages of Colletotrichum acutatum

Colletotrichum acutatum causes two diseases of citrus, postbloom fruit drop (PFD) and Key lime anthracnose (KLA). PFD is a disease restricted to flowers of sweet orange and most other citrus, and symptoms include petal necrosis, abscission of developing fruit, and the formation of persistent calyces. KLA is a disease of foliage, flowers, and fruits of Key lime only, and symptoms include necrotic lesions on leaves, fruits, twigs, flowers, and blight of entire shoots. The internal transcribed spacers 1 and 2 and the gene encoding the 5.8S ribosomal RNA subunit within the nuclear ribosomal cluster (ITS) and intron 2 of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) were sequenced for isolates from PFD-affected sweet orange and KLA-affected Key limes collected in the United States (Florida), Brazil (São Paulo), Mexico, Belize, Costa Rica, and the Dominican Republic to determine if there are consistent genetic differences between PFD and KLA isolates over the geographic area where these diseases occur. Based on the sequence data, isolates clustered into two well-supported clades with little or no sequence variation among isolates within clades. One clade (PFD clade) contained PFD isolates from all countries sampled plus a few isolates from flowers of Key lime in Brazil. The other clade (KLA clade) contained KLA isolates from Key lime foliage from all countries sampled and one isolate from flowers of sweet orange in Mexico. In greenhouse inoculations with PFD and KLA isolates from Florida, isolates from both clades produced PFD symptoms on Orlando tangelo flowers, but KLA-clade isolates produced significantly less severe symptoms. PFD-clade isolates were not pathogenic to Key lime foliage, confirming previous studies. The differentiation of PFD and KLA isolates into two well-supported clades and the pathogenicity data indicate that PFD and KLA are caused by distinct phylogenetic lineages of C. acutatum that are also biologically distinct. PFD is a recently described disease (first reported in 1979) relative to KLA (first reported in 1912) and it had been proposed that strains causing PFD evolved from strains causing KLA eventually losing pathogenicity to Key lime foliage. We reject the hypothesis that PFD strains have diverged from KLA strains recently based on estimated divergence times of haplotypes and it appears that PFD and KLA strains have been dispersed throughout the Americas independently in association with each host.

Host Range and Genetic Relatedness of Colletotrichum acutatum Isolates from Fruit Crops and Leatherleaf Fern in Florida

Isolates of Colletotrichum acutatum were collected from anthracnose-affected strawberry, leatherleaf fern, and Key lime; ripe-rot-affected blueberry; and postbloom fruit drop (PFD)-affected sweet orange in Florida. Additional isolates from ripe-rot-affected blueberry were collected from Georgia and North Carolina and from anthracnose-affected leatherleaf fern in Costa Rica. Pathogenicity tests on blueberry and strawberry fruit; foliage of Key lime, leatherleaf fern, and strawberry; and citrus flowers showed that isolates were highly pathogenic to their host of origin. Isolates were not pathogenic on foliage of heterologous hosts; however, several nonhomologous isolates were mildly or moderately pathogenic to citrus flowers and blueberry isolates were pathogenic to strawberry fruit. Based on sequence data from the internal transcribed spacer (ITS)1-5.8S rRNA-ITS2 region of the rDNA repeat, the glutaraldehyde-3-phosphate dehydrogenase intron 2 (G3PD), and the glutamine synthase intron 2 (GS), isolates from the same host were identical or very similar to each other and distinct from those isolated from other hosts. Isolates from leatherleaf fern in Florida were the only exception. Among these isolates, there were two distinct G3PD and GS sequences that occurred in three of four possible combinations. Only one of these combinations occurred in Costa Rica. Although maximum parsimony trees constructed from genomic regions individually displayed little or no homoplasy, there was a lack of concordance among genealogies that was consistent with a history of recombination. This lack of concordance was particularly evident within a clade containing PFD, Key lime, and leatherleaf fern isolates. Overall, the data indicated that it is unlikely that a pathogenic strain from one of the hosts examined would move to another of these hosts and produce an epidemic.

Effect of Fungicides and Storage Conditions on Postharvest Development of Citrus Black Spot and Survival of Guignardia citricarpa in Fruit Tissues

Citrus black spot (CBS) is caused by Guignardia citricarpa, which incites lesions on citrus fruit and can induce fruit drop. Quiescent infections occur during the spring and summer, and symptoms appear at fruit maturity or after harvest. Thus, fruit from citrus areas affected by CBS represent a risk for introduction of this pathogen into new areas. The effects of preventive field fungicide programs, postharvest fungicide drenches, packinghouse fungicide applications, and storage temperatures on postharvest symptom development and viability of G. citricarpa in lesions were evaluated in five experiments on Murcott tangor, Valencia oranges, and lemons. Preventive field treatments and fruit storage at 8°C consistently reduced postharvest CBS development, whereas a postharvest fungicide drench or packinghouse treatment with fungicides had no effect on postharvest symptom development. In a separate experiment, postharvest appearance of symptoms was related to the percentage of fruit with symptoms at harvest. The preventive field fungicide program also consistently reduced the percentage of isolation of G. citricarpa from affected fruit, whereas storage temperature and packinghouse fungicide treatment gave variable results. The viability of the fungus declined with storage time of fruit after harvest, but G. citricarpa could still be readily isolated regardless of treatment. In another experiment, the viability of the fungus in detached fruit or peel was minimally affected by temperature or moisture during storage. The frequency of successful isolation declined with time, but G. citricarpa was still recovered frequently from symptomatic tissue at later times. The most effective means to reduce postharvest development of symptoms is through preventive application of fungicides during the fruit growing season and storage of harvested fruit at cold temperatures. None of the measures evaluated substantially reduced viability of G. citricarpa, and the pathogen would likely be introduced on symptomatic fruit from citrus areas with CBS.

Selection for Pathogenicity to Strawberry in Populations of Colletotrichum gloeosporioides from Native Plants

ABSTRACT Colletotrichum gloeosporioides causes a serious crown rot of strawberry and some isolates from native plants are pathogenic to strawberry. C. gloeosporioides from lesions on wild grape and oak were sampled at two sites adjacent to commercial strawberry fields in Florida and two distant sites. Random amplified polymorphic DNA (RAPD) marker data and restriction enzyme digests of amplified rDNA were used to determine whether isolates were from the same C. gloeosporioides subgroup that infects strawberry. There were 17 to 24 native host isolates from each site that clustered with a group of strawberry crown isolates based on RAPD markers. Among strawberry isolates, there were two rDNA genotypes identified by restriction enzyme analysis. Both genotypes were present among native host isolates sampled from all four sites. There was some evidence that the different rDNA genotypes differentiated two closely related subpopulations, although the proportion of pathogenic isolates from native hosts among the two different genotypes was not different. The incidence of isolates pathogenic to strawberry was greater at sites close to strawberry fields relative to sites distant from strawberry fields for isolates with a BstUI(-)/MspI(+) rDNA genotype (44 versus 13%), a BstUI(+)/MspI(-) genotype (57 versus 16%), or when both genotypes were analyzed together (46 versus 15%). Based on these results, it appears that the C. gloeosporioides subgroup that causes crown rot on strawberry is widely distributed in Florida and that selection for pathogenicity on strawberry occurs in the area where this host is grown in abundance.

Resistance of Strawberry Cultivars to Crown Rot Caused by Colletotrichum gloeosporioides Isolates from Florida Is Nonspecific

Isolates of Colletotrichum gloeosporioides from strawberry (Fragaria × ananassa) and native grape were tested for virulence on strawberry cultivars in field experiments for three seasons. Isolate aggressiveness and cultivar resistance were determined by the proportion of plants killed at a defined time. Each year, four to six isolates were inoculated on four to seven different cultivars, with a subset of isolates and cultivars evaluated again the next season. On the dates that disease was evaluated, incidence ranged from 10 to 84% for individual cultivars. Cultivar and isolate effects were significant in all three seasons, but there was no significant cultivar by isolate interaction in any season. Thus, resistance to C. gloeosporioides appears to be nonspecific. In the third season, one isolate of Colletotrichum fragariae from strawberry and one from oak were included. There was no significant cultivar by isolate interaction detected for this species, although there were significant differences among cultivars and isolates. When the resistance of cultivars to both species was compared, the rankings of cultivars were similar, but a modest cultivar by species interaction was evident. The cultivar Treasure was more resistant to crown rot caused by either species than any other cultivar tested.

Development of a reduced use fungicide program for control of botrytis fruit rot on annual winter strawberry

Crop phenology and epidemiological information were used to design a reduced use fungicide program for control of Botrytis fruit rot in winter annual strawberry. Fungicide spray programs during early and late periods of the season using high and low rates of captan were evaluated with or without second peak bloom applications of fenhexamid during the 1999-2000 and 2000-2001 seasons. During the early harvest period, low rates of captan were as effective as high rates for controlling Botrytis fruit rot and maintaining yield. Late in the season, treatments with fenhexamid over the peak bloom period significantly improved control of Botrytis fruit rot and increased marketable yield. Application of both captan and fenhexamid during the second peak bloom did not reduce Botrytis fruit rot incidence or improve yield compared with fenhexamid alone during this time period. Late season applications of captan may be reduced or eliminated when bloom applications of fenhexamid are being applied without affecting Botrytis fruit rot control. The study generated new recommendations for use of low-rate applications of captan during the early season and applications of fenhexamid during the second peak bloom period for winter annual strawberry production in Florida.

Use of Leaf Wetness and Temperature to Time Fungicide Applications to Control Anthracnose Fruit Rot of Strawberry in Florida

Anthracnose fruit rot (AFR), caused by Colletotrichum acutatum, is a major disease of strawberry in Florida and is generally controlled by weekly fungicide applications. More than 20 applications may be made during the growing season, most commonly using captan and the quinone-outside inhibitors. Field experiments were conducted for three seasons on a susceptible and a partially resistant cultivar to evaluate the effectiveness of timing fungicide applications for managing AFR based on a previously published model by Wilson and associates that uses leaf wetness duration and temperature to predict fruit infection by C. acutatum under controlled conditions. For most treatments, rules were established where captan was applied when the predicted proportion of fruit infected (INF) from the model exceeded 0.15 and pyraclostrobin was applied when INF exceeded 0.5. For one model-timed treatment where captan and pyraclostrobin were applied before symptoms first appeared in the field, disease control was as good as the treatment where calendar weekly applications were made and the model-timed treatment utilized 47% fewer sprays. In treatments where fungicide application began after symptom appearance, the number of applications was reduced further but disease control was 40% less effective. Model-timed fungicide treatments that included pyraclostrobin gave better control than the treatments using captan alone. The model relating leaf wetness and temperature to predict AFR infection can be used effectively in a disease-forecasting system to time fungicide treatments and greatly reduce the number of applications without loss of disease control or yield.

Curative and Protectant Activity of Fungicides for Control of Crown Rot of Strawberry Caused by Colletotrichum gloeosporioides

The ability of fungicides to control Colletotrichum crown rot of strawberry caused by C. gloeosporioides was examined over three seasons. A single application of each fungicide was made 2 days before inoculation (2 DBI) or 1 day after inoculation (1 DAI) with conidial suspensions of C. gloeosporioides. The proportion of plants collapsed on one date at the end of each season was evaluated. In a combined analysis, there was a significant fungicide treatment-season interaction (P = 0.004). Percent mortality was 64% over 3 years in control plots that were inoculated with C. gloeosporioides but not treated with fungicide. Captan applied 2 DBI consistently reduced plant mortality (mean mortality = 17%). However, it was not as effective when applied 1 DAI (mean mortality = 46%). Azoxystrobin, pyraclostrobin, and thiophanate-methyl all reduced plant mortality relative to the control if applied 2 DBI (mean mortality = 46% for azoxystrobin, 37% for pyraclostrobin, and 41% for thiophanate-methyl) or 1 DAI (mean mortality = 29% for azoxystrobin, 27% for pyraclostrobin, and 32% for thiophanate-methyl). Results indicated that these fungicides were more effective when applied 1 DAI; however, lower plant mortality was not always observed with postinoculation applications. Cyprodinil + fludioxonil reduced mortality relative to the control, but there was no consistent evidence that it was more effective when applied at 2 DBI (mean mortality = 39%) than when applied 1 DAI (mean mortality = 40%). Similarly, mortality in plots treated with thiram 2 DBI (mean mortality = 30%) or 1 DAI (mean mortality = 32%) was not different. Potassium phosphite did not affect mortality, regardless of the timing of application (2 DBI mean mortality = 61%, 1 DAI mean mortality = 67%). The results indicated that an effective strategy for controlling Colletotrichum crown rot caused by C. gloeosporioides should be based on weekly applications of captan throughout the growing season. Azoxystrobin, pyraclostrobin, or thiophanate-methyl applications should be applied when weather conditions are highly favorable for disease development and the activity of contact fungicides such as captan or thiram might be compromised.