Colletotrichum caladii sp. nov. Causing Anthracnose Leaf Spot of Caladium × hortulanum (Araceae) in Florida, U.S.A.

Caladium (Caladium × hortulanum) is an ornamental plant popular for its variable and colorful foliage. In 2020, plants showing leaf spots and blight, typical of anthracnose, were found in a field trial at the University of Florida's Gulf Coast Research and Education Center in Wimauma, Florida, U.S.A. Leaf samples consistently yielded a Colletotrichum-like species with curved conidia and abundant setae production in the acervuli. The internal transcribed spacer (ITS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase gene (gapdh), actin gene (act), chitin synthase 1 gene (chs-1), beta-tubulin gene (tub2), and histone3 gene (his3) were amplified and sequenced. BLASTN searches in the NCBI GenBank database revealed similarities to species of the Colletotrichum truncatum species complex. Phylogenetic analyses using multilocus sequence data supports a distinct species within this complex, with the closest related species being C. curcumae. Based on morphological and phylogenetic analyses, a new species of Colletotrichum, named C. caladii, is reported. Pathogenicity assays and subsequent isolation confirmed that this species was the causal agent of the disease.

Resistance of Strawberry Cultivars and the Effects of Plant Ontogenesis on Phytophthora cactorum and P. nicotianae Causing Crown Rot.

Phytophthora crown rot (PhCR) is an important disease of strawberry worldwide. Phytophthora cactorum is the most common causal agent, however, P. nicotianae was also recently reported causing PhCR in the U.S. Therefore, the goals of this study were to evaluate the resistance of strawberry cultivars from Florida and California, and to study the etiology of the two Phytophthora species causing PhCR. Sixteen strawberry cultivars were evaluated over three Florida seasons for susceptibility to P. cactorum, and P. nicotianae. Inoculations at different days after transplanting (DAT) were also carried out to evaluate the ability of both species to cause PhCR at different phenological stages of the plant. Plant wilting and mortality were assessed weekly, and disease incidence, and the area under the disease progress curve were calculated. Cultivars Sensation 'Florida127', 'Winterstar FL 05-107', and 'Florida Radiance' were susceptible, whereas 'Florida Elyana', 'Camarosa', 'Fronteras', 'Sweet Charlie', and 'Strawberry Festival' were highly resistant to both Phytophthora species. However, some cultivars exhibited stronger resistance to one species over the other. P. cactorum caused more PhCR when plants were inoculated at transplanting, 45, and 60 DAT, whereas P. nicotianae only caused disease when inoculated at transplanting. These results emphasize the importance of screening for disease resistance to guide management recommendations in commercial strawberry production as well as the need for proper pathogen identification since cultivar susceptibility might differ. Varying susceptibility to P. cactorum and P. nicotianae at different growth stages emphasizes the importance of considering both plant and pathogen biology when making management recommendations.

Pseudocercospora pancratii Causing Leaf Spots on Commercial Blackberry (Rubus sp.) in Florida.

Blackberry (Rubus L. subgenus Rubus Watson) is a deciduous berry crop that is the fourth most economically important berry crop, and its production is expanding in the southeastern United States. However, since most commercially available cultivars were bred under temperate conditions, they are not always well adapted and could be threatened by new pathogen populations inhabiting subtropical areas. In 2017, plants showing purple or brown leaf spots and angular-to-irregular lesions on both leaf surfaces, with clusters of black conidiophores at the center, were observed in a field trial at the University of Florida's Gulf Coast Research and Education Center (UF/GCREC) in Wimauma, FL. A fungus resembling Cercospora/Pseudocercospora was isolated from the lesions. The ribosomal DNA internal transcribed spacers, the translation elongation factor 1-alpha, and the actin genes were amplified and sequenced. Based on the phylogenetic analysis, the closest related species was Pseudocercospora pancratii. Pathogenicity assays and subsequent reisolation confirmed that this species is the causal agent of the disease. Among eight cultivars screened, no complete resistance was found. However, 'Osage' was the least susceptible, and 'Kiowa' was the most susceptible. This study is the first report of P. pancratii causing leaf spots on blackberry worldwide, and it may help shape future research into disease epidemiology and management for a crop that is rapidly expanding but has very limited disease information currently available for Florida growers.

Detection and Characterization of Quinone Outside Inhibitor-Resistant Phytophthora cactorum and P. nicotianae Causing Leather Rot in Florida Strawberry.

Phytophthora cactorum and P. nicotianae cause leather rot (LR) of fruit and Phytophthora crown rot (PhCR) in strawberry. LR occurs sporadically but can cause up to 70% fruit loss when weather is conducive. In Florida's annual strawberry winter production system, PhCR can be severe, resulting in plant stunting, mortality, and severe yield loss. Azoxystrobin is labeled for control of LR but not for PhCR. The aims of this research were to determine the sensitivity of P. cactorum and P. nicotianae isolates from strawberry to azoxystrobin and to investigate mechanisms of quinone-outside-inhibitor resistance present in P. cactorum and P. nicotianae based on the known point mutations within the cytochrome b (cytb) gene. Isolates of both Phytophthora spp. causing LR and PhCR were collected from multiple strawberry fields in Florida between 1997 and 2020. Isolates were tested for sensitivity to azoxystrobin at 0, 0.01, 0.1, 1.0, 10, and 50 µg/ml on potato dextrose agar amended with salicylhydroxamic acid (100 µg/ml). Isolates were separated into two groups - sensitive isolates with the 50% effective concentration (EC50) values <1.0 µg/ml, and resistant isolates having EC50 values >50 µg/ml. P. cactorum and P. nicotianae resistance to azoxystrobin was found for isolates collected after 2010. The first 450 nucleotides of the mitochondrial cytb gene were sequenced from a selection of resistant and sensitive isolates of both species. The G143A mutation reported to confer resistance to azoxystrobin was found in all resistant P. cactorum isolates. However, in P. nicotianae, qualitative resistance was observed, but the isolates lacked all the known mutations in the cytb gene. This is the first report of resistance to azoxystrobin in P. cactorum and P. nicotianae.

First Report of Leaf Rust on Blackberry (Rubus spp.) Caused by Kuehneola uredinis in Florida.

During the fall of 2020 and summer of 2021, symptoms of leaf rust were observed on blackberry plants of 'Kiowa', and breeding line 1734 (progeny of 'Natchez' and Arapaho') in a field trial at the University of Florida, Wimauma, FL. Symptoms consisted of small chlorotic spots (1 to 3 mm) on the upper side of the leaf, while the underside had yellow-orange pustules. Disease incidence was up to 100% on both 'Kiowa' and the breeding line 1734, and severity was up to 20% with most of the symptoms observed on older leaves. Two isolates were collected from 'Kiowa' and one from the breeding line 1734 for further investigation. Isolates were maintained and multiplied on healthy 'Kiowa' plants in growth chambers (25 ºC and 12-12 h photoperiod). Uredinia (n=30) were erumpent and ranged from 90 to 320 µm (Average=285 µm, SD=5.3 µm) in diameter. Urediniospores (n=50) were obovoid, yellow, and ranged from 24 to 36 µm long (Average=32 µm, SD=3.2 µm) and 22 to 30 µm wide (Average=28 µm, SD=2.5 µm). Based on morphology and literature, the pathogen was tentatively identified as Kuehneola uredinis (Link) Arth (Arthur 1906; Shands et al., 2018). Spores from a single uredinium of each isolate were collected with a needle and suspended in 50 µL of molecular biology-grade water yielding a final concentration of approximately 5 x 104 spores/mL. Two µL of each spore suspension was used for the PCR reactions. Two DNA fragments were amplified using the primers Rust2inv and LR6, and Rust18S-R and NS1 for the 5.8S-ITS2-28S gene region of rDNA (1,755 bp) and partial 18S gene region of rDNA (2,684 bp), respectively. The amplified products of the partial 28S gene region were sequenced with the primers LR3 and LR0R, and the 18S gene region with NS5, NS6, and NS4 (Aime 2006). DNA sequences were deposited in GenBank (accession nos. OK509845 - OK509848). BLASTn searches revealed that the isolates were 100% identical to K. uredinis reported causing leaf rust on blackberry in California (1044/1044bp, and 1540/1540bp for accession numbers MF158087, and MF158088, respectively). To test for pathogenicity, blackberry cultivars Kiowa, Natchez, Osage, Ouachita, Ponca, Prime-Ark® 45, Prime-Ark® Freedom, Prime-Ark® Traveler, and Prime-Ark® Horizon were inoculated. Five plants of each cultivar were inoculated with a mixture of spores of the three isolates, and two plants of each cultivar were used as controls. Spores were washed from leaves of 'Kiowa' exhibiting sporulation using a suspension of 1% Tween 20 in deionized water. The final concentration of the inoculum was adjusted to 104 spores/mL. Plants were inoculated in the greenhouse with a spray bottle until run-off and kept inside clear plastic boxes for 48 h. Controls were sprayed with sterile deionized water. Plants were watered by mists of 3 s every 10 min twice a week. Disease incidence and severity were evaluated weekly on five leaves per plant that had been tagged before inoculation. The experiment was repeated once. Symptoms identical to the original were only observed in 'Kiowa' and 'Prime-Ark® Freedom'. One week after inoculation, disease incidence was already 100% in both cultivars, with at least one pustule on all the inoculated leaves, and six weeks later disease severity was up to 50% (Average= 35%, SD=2.4%). To our knowledge, this is the first report of K. uredinis causing leaf rust on blackberry in Florida. This disease was reported on Rubus spp. in several U.S. states, and recently in California on Rubus ursinus (Farr and Rossman 2021; Shands et al. 2018). Blackberry is an emerging crop in Florida and efforts should be implemented to monitor the occurrence and spread of leaf rust considering that urediniospores disperse long distances by wind, especially if growers choose the susceptible cultivars 'Kiowa' and 'Prime-Ark Freedom'. The apparent resistance observed in other commercial cultivars such as 'Osage', 'Ouachita', and 'Ponca' may serve as valuable breeding parents for developing new blackberry cultivars with resistance to leaf rust.

Resistance to Mefenoxam of Phytophthora cactorum and Phytophthora nicotianae Causing Crown and Leather Rot in Florida Strawberry.

Phytophthora cactorum and Phytophthora nicotianae cause leather rot on fruit and crown rot (PhCR) of strawberry plants. Leather rot is not a common disease in Florida; however, up to 50% yield loss has been reported in harvests after intense rainfall events. PhCR is an important disease worldwide and is characterized by a sudden wilting and collapse of plants. Mefenoxam is the most effective and widely used fungicide to control both diseases. P. cactorum and P. nicotianae isolates from leather rot and PhCR have been collected from multiple strawberry fields in Florida since 1997, and the sensitivity of 185 isolates was tested at 0, 0.05, 0.5, 5, and 100 µg/ml. The 50% effective concentration (EC50) values of sensitive isolates ranged from 0.05 to 1 µg/ml. Resistance to mefenoxam (EC50 values >100 µg/ml) was found among P. cactorum isolates collected after 2015, but no resistance was found in P. nicotianae isolates. During the 2015 to 2016, 2016 to 2017, 2017 to 2018, and 2018 to 2019 seasons, resistance was detected on 9, 10, 21, and 23% of the isolates collected, respectively. Mefenoxam-resistant isolates originated from 3 of the 24 strawberry nurseries monitored. This is the first report of the occurrence of P. cactorum resistance to mefenoxam in Florida, suggesting that alternative control strategies are needed to avoid the increase of mefenoxam-resistant populations of P. cactorum in Florida fields.

Cultivar Selection Is an Effective and Economic Strategy for Managing Charcoal Rot of Strawberry in Florida.

Macrophomina phaseolina, the causal agent of charcoal rot, is a soilborne pathogen that affects strawberry crowns leading to plant wilt and collapse. Disease management involves a combination of physical, cultural, and chemical methods. Field trials were conducted for 10 consecutive Florida seasons (2010-11 to 2019-20) to determine the susceptibility of strawberry cultivars to charcoal rot and the effect of cultivar selection on disease and to estimate the economic impact of cultivar selection on disease management. Six cultivars grown commercially in Florida were chosen and grouped as highly susceptible (HS) ('Strawberry Festival' and 'Treasure'), susceptible (S) ('Florida Radiance' and 'Florida Beauty'), and moderately resistant (MR) (Sensation 'Florida127' and Winterstar 'FL05-107') according to their susceptibility levels. After a primary analysis of the individual trials, a network meta-analysis was conducted to estimate and compare the final disease incidence and the disease progress rate of each susceptibility group. The economic impact of charcoal rot on strawberry production and gross revenue was estimated based on plant production functions, weekly fruit prices, and disease progress over time with parameters obtained via the meta-analytical models. Disease incidence was reduced by 91.5 and 77.3%, respectively, when the MR and S cultivar groups were adopted instead of the HS group. There was a 62.5% reduction in the disease incidence when the MR group was used instead of the S group. Significant differences in disease progress rates were also observed when the MR and S groups were adopted instead of the HS group. Therefore, the adoption of more resistant cultivars is an effective strategy when incorporated into a charcoal rot integrated management program and can significantly impact growers' revenue by reducing disease incidence, preventing yield loss, and, consequently, minimizing economic losses.

Resistance in Strawberry Isolates of Colletotrichum acutatum from Florida to Quinone-Outside Inhibitor Fungicides.

Anthracnose fruit rot of strawberry, caused by Colletotrichum acutatum, is a major disease in Florida and frequent quinone-outside inhibitor (QoI) fungicide applications are needed for disease control. From 1994 to 2014, 181 C. acutatum isolates were collected from multiple strawberry fields in Florida with or without QoI spray history. Sensitivity to azoxystrobin and pyraclostrobin was tested based upon mycelial growth and germ tube elongation inhibition. Mean effective concentration where growth was reduced by 50% (EC50) values for isolates collected prior to 2013 based upon mycelial growth were 0.22 and 0.013 µg/ml and upon germ tube elongation were 0.57 and 0.03 µg/ml for azoxystrobin and pyraclostrobin, respectively. Mycelial growth and germ tube elongation of 48 isolates collected in 2013 and 2014 were not inhibited with azoxystrobin at 3 µg/ml and pyraclostrobin at 0.110 µg/ml. A fungicide discriminatory dose assay indicated that 43 of the 48 isolates had EC50 values higher than 100 and 10 µg/ml for azoxystrobin and pyraclostrobin, respectively. Azoxystrobin and pyraclostrobin sprayed preventively on strawberry fruit inoculated with C. acutatum failed to control resistant isolates. Sequencing of the cytochrome b gene of sensitive and resistant isolates showed that QoI-resistant isolates contained either G143A or F129L amino acid substitutions.

Evaluating Weeds as Hosts of Tomato yellow leaf curl virus.

Bemisia tabaci (Gennadius) biotype B transmits Tomato yellow leaf curl virus (TYLCV), which affects tomato production globally. Prompt destruction of virus reservoirs is a key component of virus management. Identification of weed hosts of TYLCV will be useful for reducing such reservoirs. The status of weeds as alternate hosts of TYLCV in Florida remains unclear. In greenhouse studies, B. tabaci adults from a colony reared on TYLCV-infected tomato were established in cages containing one of four weeds common to horticultural fields in central and south Florida. Cages containing tomato and cotton were also infested with viruliferous whiteflies as a positive control and negative control, respectively. Whitefly adults and plant tissue were tested periodically over 10 wk for the presence of TYLCV using PCR. After 10 wk, virus-susceptible tomato plants were placed in each cage to determine if whiteflies descended from the original adults were still infective. Results indicate that Bidens alba, Emilia fosbergii, and Raphanus raphanistrum are not hosts of TYLCV, and that Amaranthus retroflexus is a host.