Natalia A. Peres

Benomyl sensitivity of isolates of Colletotrichum acutatum and C. gloeosporioides from citrus

Postbloom fruit drop (PFD) of citrus, caused by Colletotrichum acutatum, produces orange-brown lesions on petals and results in premature fruit drop and the retention of calyces. C. gloeosporioides is common in groves and causes postharvest anthracnose on fruit. Both diseases are controlled effectively by the fungicide benomyl in research fields and commercial orchards. Highly sensitive and resistant isolates of C. gloeosporioides were found, whereas all isolates of C. acutatum tested were moderately resistant. In preliminary studies conducted in vitro with three isolates of each, mycelial growth of sensitive isolates of C. gloeosporioides was inhibited completely by benomyl (Benlate 50 WP) at 1.0 μg/ml, whereas resistant isolates grew well at 10 μg/ml. Growth of all isolates of C. acutatum was inhibited by about 55% at 0.1 μg/ml and by 80% at 1.0 μg/ml. Spore germination of C. acutatum was inhibited more at 0.1 μg/ml than at 1.0 μg/ml or higher concentrations. In all, 20 isolates of C. acutatum from 17 groves and 20 isolates of C. gloeosporioides from 7 groves were collected from locations with different histories of benomyl usage in São Paulo, Brazil, and Florida, United States. Benomyl at 1.0 μg/ml completely inhibited growth of 133 isolates of C. gloeosporioides, with the exception of 7 isolates that were highly resistant to the fungicide, whereas all isolates of C. acutatum were only partially inhibited at 0.1 and 1.0 μg/ml. Analysis of variance indicated that the sensitivity of the isolates of C. acutatum was not affected by benomyl usage or grove of origin, and country of origin had only minor effects. No highly resistant or sensitive isolate of C. acutatum was recovered. Partial sequencing of the β-tubulin gene did not reveal nucleotide substitutions in codons 198 or 200 in C. acutatum that usually are associated with benomyl resistance in other fungi.

Phenotypic Characterization of Multifungicide Resistance in Botrytis cinerea Isolates from Strawberry Fields in Florida

Chemical control has always been essential for the management of gray mold, caused by Botrytis cinerea, to ensure sustainable strawberry production. However, lack of knowledge about actual resistance development may have disastrous consequences and lead to severe epidemics such as the one that affected several strawberry fields in 2012 in Florida. In this study, we tested 392 isolates collected from Florida strawberry fields between 2010 and 2012 for their sensitivity to boscalid (Bosc), a succinate dehydrogenase inhibitor (SdhI); pyraclostrobin, a quinone outside inhibitor (QoI); boscalid + pyraclostrobin (Pristine); fenhexamid, a hydroxyanilide (Hyd); pyrimethanil and cyprodinil, anilinopyrimidines; fludioxonil, a phenylpyrrole; and fludioxonil + cyprodinil (Switch). The respective resistance frequencies for boscalid, pyraclostrobin, Pristine, fenhexamid, cyprodinil, and pyrimethanil were 85.4, 86.5, 86.0, 44.4, 52.7, and 59.5%. Overall, 17.8 and 19.8% of isolates showed reduced sensitivity to fludioxonil and Switch, respectively. All fungicides sprayed preventively on detached strawberry fruit failed to control isolates with high levels of resistance to each fungicide except for fludioxonil and Switch. Four phenotypes with multifungicide resistance (MFR) were detected in B. cinerea populations from Florida. Isolates resistant to one fungicide (FR1), two (MFR2), three (MFR3), and four (MFR4) fungicides from different chemical groups represented 5.9, 28.6, 41.8, and 23.7% of the total resistant population, respectively. The MFR3 isolates were predominant and contained two subpopulations, the Bosc-QoI-APR isolates (56.5%) and the Bosc-QoI-HydR isolates (40.6%). In addition to reporting on very highly resistant populations to boscalid and QoI fungicides, we show evidence for a widespread multifungicide resistance to B. cinerea that warrants immediate implementation of novel management strategies to impede the development of more resistant populations.

Resistance to Fluopyram, Fluxapyroxad, and Penthiopyrad in Botrytis cinerea from Strawberry

Succinate dehydrogenase inhibitors (SDHIs) constitute a mainstay in management of gray mold caused by Botrytis cinerea in strawberry and several other crops. In this study, we investigated the risks of resistance development to three newer SDHIs (i.e., fluopyram, fluxapyroxad, and penthiopyrad) and their cross-resistance with the previously registered boscalid. We investigated the mutations in the SdhB subunit and evaluated their impact on microbial fitness in field populations of B. cinerea. Amino acid substitutions associated with resistance to SDHIs were detected at three codons of the SdhB subunit (BH272R/Y/L, BP225F, and BN230I) in the succinate dehydrogenase gene of field isolates from Florida. The BH272R, BH272Y, BH272L, BP225F, and BN230I mutations were detected at frequencies of 51.5, 28.0, 0.5, 2.5, and 4%, respectively. Strong cross-resistance patterns were evident between boscalid and fluxapyroxad and penthiopyrad but not with fluopyram, except in BH272L, BP225F, and BN230I mutants. All five mutations conferred moderate to very high resistance to boscalid whereas the BH272Y conferred resistance to fluxapyroxad and penthiopyrad. The BH272L, BN230I, and BP225F mutations conferred high resistance to all four SDHIs tested. Resistance monitoring following the first use of penthiopyrad in strawberry fields in Florida in 2013 suggests potential for quick selection for highly resistant populations and warrants careful use of the newer SDHIs. No evidence of major fitness costs due to the mutations in the SdhB subunit was found, which indicates the potential ability of the mutants to survive and compete with wild-type isolates. Our study suggests high risks for rapid widespread occurrence of B. cinerea populations resistant to the novel SDHIs unless appropriate rotation strategies are implemented immediately upon registration.

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.

Comparison of Molecular Procedures for Detection and Identification of Guignardia citricarpa and G. mangiferae

Citrus black spot, caused by Guignardia citricarpa, is a serious fruit spot disease and is widely distributed in Asia, southern Africa, and South America, but does not occur in North America or the Mediterranean region. A nonpathogenic species, G. mangiferae, is cosmopolitan with a wide host range and can colonize citrus fruit and leaves saprophytically. Detection and identification of Guignardia spp. on citrus fruit is necessary for epidemiological, management, and regulatory purposes. In this study, we compared published and unpublished polymerase chain reaction primer sets for their specificity and sensitivity in the detection and differentiation of the two Guignardia spp. All primers evaluated successfully identified the two species using purified DNA from fungal cultures or mycelia as source materials. However, some primer sets were not highly effective in detecting G. citricarpa when DNA was extracted directly from single characteristic black spot lesions on fruit. Thus, new primer pairs for both species were designed from the internal transcribed spacer region that were highly sensitive and specific for detection of G. citricarpa using DNA recovered from single lesions on fruit by a rapid DNA extraction procedure.

Fungicide Resistance Profiles in Botrytis cinerea from Strawberry Fields of Seven Southern U.S. States

The sensitivity to seven chemical classes of fungicides was investigated in 1,810 Botrytis cinerea isolates collected from strawberry blossoms and fruit in 181 strawberry fields from seven southern states in the United States across 2 years. Ten isolates were examined from each field. Fungicide sensitivity assays were carried out based on visual assessment of diametrical mycelial growth after 4 days of incubation on media amended with discriminatory doses of fungicides in microtiter plates. Results of visual assessments were verified with selected isolates using a previously published germination assay and by inoculating representative isolates with resistant phenotypes on fungicide-sprayed fruit. The overall resistance frequencies of 750 isolates collected in 2012 for thiophanate-methyl, pyraclostrobin, boscalid, cyprodinil, fenhexamid, iprodione, and fludioxonil were 76, 42, 29, 27, 25, 3, and 1%, respectively. Frequencies of 1,060 isolates collected in 2013 were 85, 59, 5, 17, 26, 2, and 1%, respectively. Resistance to thiophanate-methyl and pyraclostrobin was found in virtually every location in both years, whereas resistance to iprodione and fludioxonil was rarely found. Resistant isolates were resistant to either one (23%), two (18%), three (19%), four (14%), five (3%), or six (0.1%) chemical classes of fungicides in 2012. In 2013, this distribution was 24, 29, 26, 8, 2, and 0.3%, respectively. Multifungicide-resistant isolates of B. cinerea were widespread in southern states and evidence suggests that the frequency of isolates with multifungicide resistance increased from 2012 to 2013. The data also show that fungicide resistance in B. cinerea was already present in blossoms, indicating that resistance management needs to be implemented early in the season.

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.

Characterization of Iprodione Resistance in Botrytis cinerea from Strawberry and Blackberry

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of strawberry. Control of the disease in commercial fields is largely dependent on the application of fungicides, including the dicarboximide iprodione. Single-spore isolates were collected from strawberry fields in Florida, North Carolina, and South Carolina and subjected to an assay using conidial germination that distinguished sensitive (S) isolates from isolates with various levels of resistance to iprodione. Of the 245 isolates, 1 was highly resistant (HR), 5 were moderately resistant (MR), and 43 had low resistance (LR) to iprodione. LR and MR strains were found in the Florida population and in 9 of 11 locations from North Carolina and South Carolina, indicating that resistance was widespread but accounted for only a relatively small percentage of the B. cinerea population. Sequence analysis of the target gene bos1, which codes for a class III histidine kinase, revealed that the MR phenotype was associated with Q369P and N373S mutations and that the LR phenotype was associated with either a I365S or a I365N mutation. The I365S and I365N mutations were also present in five additionally included HR isolates from North Carolina and South Carolina blackberry fields and one HR isolate from a Virginia strawberry field but no mutation or mutation combinations in bos1 were uniquely associated with the HR phenotype. Expression analysis of bos1 in S and HR isolates did not reveal convincing evidence of the genes involvement in HR resistance either. The six HR isolates had three different phenotypes with respect to their sensitivity to fludioxonil; two were S, two were LR, and two were MR. The fludioxonil LR and MR isolates were also resistant to tolnaftate, an indication of multidrug efflux pump activity. These data suggest that, in addition to point mutations in bos1, drug efflux pump activity and potentially a third mechanism of resistance may be contributing to the iprodione HR phenotype. Detached fruit studies showed that field rates of Rovral 4 Flowable (iprodione) did not control iprodione MR and HR isolates.

Pre- and Post-Infection Activity of Pyraclostrobin for Control of Anthracnose Fruit Rot of Strawberry Caused by Colletotrichum acutatum

The effect of pre- and post-infection-period applications of pyraclostrobin (Cabrio EG) on the development of anthracnose fruit rot was characterized in a controlled-climate study and validated in field studies in New York and Florida. Plants of the day-neutral cv. Tristar were inoculated with C. acutatum and placed into mist chambers at 14, 22, or 30°C. The plants were removed from the chambers after 3, 6, 12, or 24 h of misting and placed on greenhouse benches to allow disease development. The fungicide pyraclostrobin was applied to the berries at a concentration equivalent to 168 g a.i./ha at 3, 8, 24, and 48 h prior to inoculation and exposure to their wetting period, or 3, 8, 24, and 48 h following inoculation and exposure to their wetting period. All pyraclostrobin treatments suppressed disease compared with the corresponding untreated control treatments. The highest incidence of disease occurred on plants exposed to the longest wetness durations (12 and 24 h) or highest temperature treatments (22 and 30°C). Post-infection applications of pyraclostrobin provided significant control when applications were made within 3 and often up to 8 h after wetting, but generally were less effective than protective sprays. We further tested the ability of pyraclostrobin to control anthracnose when applied as a protectant or as an after-infection application in inoculated field plots exposed to a short (8 h) or long (24 h) wetting period in Florida and in New York. In three of the four experimental plots, disease control equivalent to or better than the protective spray was achieved when pyraclostrobin was applied up to 24 h after infection for long and short wetting periods. In the remaining plot, conditions for disease development were exceptionally favorable. The protective treatment provided approximately 75% control, whereas the best post-infection treatment provided only 50% control. Our study indicates that for short wetting events, such as those associated with seasonal thunderstorms, growers can wait until after such an infection event before applying pyraclos-trobin and achieve control equivalent to a protective application.

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.