Assessing Biogeography of Coffee Rust Risk in Brazil as Affected by the El Niño Southern Oscillation.

The El Niño Southern Oscillation (ENSO) is an oceanic-atmospheric phenomenon influencing worldwide weather and climate. Its occurrence is determined by the sea surface temperature (SST) anomaly of the 3.4 Niño region in the Pacific Ocean (5°N-5°S, 120°-170°W). El Niño (EN), Neutral (NT), and La Niña (LN) are the three possible phases of ENSO, respectively, for warm, normal, and cold SST anomaly. As in other regions around the world, weather in Brazil is influenced by ENSO phases. The country is the major coffee producer in the world, and production is strongly influenced by weather conditions, which affect plant yield, harvest quality, and interactions with pests and diseases. Coffee leaf rust (CLR), caused by the fungus Hemileia vastatrix, is a major cause of coffee yield and quality losses in Brazil, and requires fungicide spray applications every season. Because CLR is highly influenced by weather conditions, it is possible to use weather variables to simulate its progress during the cropping cycle. Therefore, the aims of this study were to estimate CLR infection rate based on a validated empirical model, which has daily minimum air temperature and relative humidity as inputs, and to assess the extent of ENSO influence on the annual risk of this disease at 45 sites in Brazil. Cumulative infection rates (CIR) were estimated daily from October to June of each growing season and location, based on the prevailing ENSO phase. Differences between the extreme phases (EN-LN) were assessed by the Two-One-Sided-Tests (TOST) method. Analysis of data from eight sites, located mainly in Paraná State, provided evidence of CIR differences between EN and LN phases (G1). Evidence of no difference of CIR between EN and LN was found in 18 sites (G2), whereas 19 sites showed no evidence of differences (G3) due to relatively large variation of CIR within the same ENSO phase. The G1 sites are located mostly in Southern Brazil, where ENSO exerts a well-defined influence on rainfall regime. In contrast, the G2 sites are mainly in Minas Gerais State, which is characterized as a transition region for ENSO influence on rainfall. The G3 sites are located between the northern region of Minas Gerais State and southern region of Bahia State, which is characterized by a subhumid climate that is usually very dry during winter, and where rainfall can vary up to 300% from one year to another, influencing relative humidity and resulting in a high CIR variability. Therefore, ENSO had a well-defined influence on CIR only in Paraná State, a region with minor importance for coffee production in Brazil. No ENSO influence was found in more northerly zones where the majority of Brazilian coffee is produced. This is the first evidence of ENSO-linked regional impact on the risk of coffee rust.

Genera Acremonium and Sarocladium Cause Brown Spot on Bagged Apple Fruit in China.

Apple fruit spot disease has caused serious economic losses for years in China since the widespread application of fruit bagging in production. Although the three genera Trichothecium, Alternaria, and Acremonium have been reported to be the causal agents, studies on the disease etiology and pathogen biology are still sparse. Here, we report characterization of eight fungal isolates from lesions on 126 symptomatic fruit samples collected in Shaanxi Province, China. Pathogenicity of the isolates was assessed. DNA sequences were obtained at four loci, including D1/D2 domains of the large-subunit nrRNA gene, internal transcribed spacer regions 1 and 2, 5.8S nrDAN gene, a fragment of the actin gene, and a fragment of the ß-tubulin. Based on phylogenetic analysis and morphological features, three new species were found: Acremonium mali, Sarocladium liquanensis, and Sarocladium mali. In addition, we made the first report of Sarocladium terricola as a plant pathogen. Temperature and moisture significantly affected in vitro conidial germination of five Acremonium-like species, and their impact on infection of apple fruit was tested using Acremonium sclerotigenum. Conidia of five species germinated from 15 to 35°C in free water; four of the species had optimum temperature around 25°C, whereas conidia of S. terricola had an optimum temperature of 30°C. Conidial germination rate increased as relative humidity (RH) increased. The five isolates had relatively high conidial germination rates at RH > 97%, with a significant decline at 95% RH. Incidence of infection also increased in proportion to RH. In free water, conidial germination was relatively unaffected by temperature.

Alternaria malicola sp. nov., a New Pathogen Causing Fruit Spot on Apple in China.

Alternaria spp. are pathogens of several diseases that pose significant threats to apple production. Several putative Alternaria sp. isolates were obtained from lesions of a disease commonly referred to as black dot on apple fruit in Shaanxi Province, China. Pathogenicity tests using mycelial plugs and conidial suspensions indicated that this isolate could cause leaf blotch, as well as moldy core and black dot on fruit. On the basis of sequence analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit, and translation elongation factor 1-α, an isolate clustered with the Alternaria sect. Ulocladioides. By combining GAPDH, major allergen Alta1, mating type protein 1-2-1, and the AGA1 gene sequence analysis and morphological description, the isolates were identified as a new species named Alternaria malicola. Our finding expands the documented diversity of apple pathogens within the genus Alternaria and clarifies the taxonomy of the pathogen assemblage that may be associated with three apple diseases.

Effects of Fungicides and Spray Application Interval on Controlling Marssonina Blotch of Apple in the Loess Plateau Region of China.

Marssonina blotch, caused by the fungus Marssonina coronariae, is a serious foliar disease on apple in East Asia as well as in other moist temperate regions in Asia, Europe, and South America. Several fungicides were investigated for their toxicity to mycelial growth and conidial germination of the pathogen in vitro. Tebuconazole, kresoxim-methyl, hexaconazole, propiconazole, and a mixture of tebuconazole and benziothiazolinone sharply inhibited mycelial growth but had less effect on conidial germination. Field tests were conducted in a commercial orchard in Baishui County, Shaanxi Province, China, during 2012, 2013, 2014, and 2015 in order to develop recommendations for apple growers. Three applications of tebuconazole, hexaconazole, propiconazole, or a mixture of tebuconazole and benziothiazolinone at 20-day intervals from early July to late August resulted in defoliation incidence of <5%. When sprays of Bordeaux mixture + tebuconazole, Bordeaux mixture + propiconazole, and Bordeaux mixture + tebuconazole and benziothiazolinone were alternated, the spray interval was extended to 25 days and defoliation incidence remained <5%. Based on historical records and our results, scouting for symptoms should begin in mid-June. We recommend commencing the spray period in early July in years with normal rainfall patterns, and spraying in mid- to late June in years with much rainfall. The findings of this study create a foundation for implementation of an efficient spray program against Marssonina leaf blotch in apple orchards in the Loess Plateau Region of China.

Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification.

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the ß-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml-1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml-1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml-1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

Management of Valsa Canker on Apple with Adjustments to Potassium Nutrition.

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple in the primary production areas of China and other East Asian countries. Currently, there are no effective control methods for this disease. We investigated the occurrence of Valsa canker in 24 apple orchards in Shaanxi Province in concert with foliar nutrient analysis, and found that there was a significant negative correlation of leaf potassium (K) content with incidence and severity of Valsa canker. Fertilization experiments showed that increasing tree K content enhanced resistance to pathogen colonization and establishment. Apple trees with leaf K content greater than 1.30% exhibited almost complete resistance to Valsa mali. Field trials demonstrated that increasing K fertilization could significantly reduce disease incidence. Improved management of tree nutrition, especially K content, could effectively control the occurrence and development of Valsa canker.

Phenology of Infection on Apple Fruit by Sooty Blotch and Flyspeck Species in Iowa Apple Orchards.

Sooty blotch and flyspeck (SBFS) is a fungal disease complex that can cause significant economic losses to apple growers by blemishing the fruit surface with dark-colored colonies. Little is known about the phenology of host infection for this diverse group of epiphytes. In 2009 and 2010, we investigated the timing of infection of apple fruit by SBFS species in six commercial apple orchards in Iowa. Five trees in each orchard received no fungicide sprays after fruit set. Within 3 weeks after fruit set, 60 apples per tree were covered with Japanese fruit bags to minimize inoculum deposition. Subsequently, a subsample of bagged apples was exposed for a single 2-week-long period and then rebagged for the remainder of the growing season. Experimental treatments included seven consecutive 2-week-long exposure periods; control treatments were apples that were either bagged or exposed for the entire season. After apples had been stored at 2°C for 6 weeks following harvest, all SBFS colonies on the apples were identified to species using a PCR-RFLP protocol. A total of 15 species were identified. For the seven most prevalent species, the number of infections per cm2 of fruit surface was greatest on apples that had been exposed early in the season. Two SBFS species, Peltaster fructicola and Colletogloeopsis-like FG2, differed significantly from each other in time required to attain 50% of the total number of colonies per apple, and analysis of variance indicated a significant interaction of SBFS taxon with exposure period. Our findings are the first evidence of species-specific patterns in timing of SBFS inoculum deposition and infection on apple fruit, and strengthen previous observations that most SBFS infections resulting in visible colonies at harvest develop from infections that occur early in the fruit development period. By defining taxon-specific phenological patterns of fruit infection, our findings, when combined with knowledge of region-specific patterns of taxon prevalence, provide a foundation for development of more efficient and cost-effective SBFS management tactics.

Genetic and virulence variability among Erwinia tracheiphila strains recovered from different cucurbit hosts.

The causal agent of cucurbit bacterial wilt, Erwinia tracheiphila, has a wide host range in the family Cucurbitaceae, including economically important crops such as muskmelon (Cucumis melo), cucumber (C. sativus), and squash (Cucurbita spp.). Genetic variability of 69 E. tracheiphila strains was investigated by repetitive-element polymerase chain reaction (rep-PCR) using BOXA1R and ERIC1-2 primers. Fingerprint profiles revealed significant variability associated with crop host; strains isolated from Cucumis spp. were clearly distinguishable from Cucurbita spp.-isolated strains regardless of geographic origin. Twelve E. tracheiphila strains isolated from muskmelon, cucumber, or summer squash were inoculated onto muskmelon and summer squash seedlings, followed by incubation in a growth chamber. Wilt symptoms were assessed over 3 weeks, strains were reisolated, and rep-PCR profiles were compared with the inoculated strains. Wilting occurred significantly faster when seedlings were inoculated with strains that originated from the same crop host genus (P<0.001). In the first run of the experiment, cucumber and muskmelon strains caused wilting on muskmelon seedlings at a median of 7.8 and 5.6 days after inoculation (dai), respectively. Summer squash seedlings wilted 18.0, 15.7, and 5.7 dai when inoculated with muskmelon-, cucumber-, and squash-origin strains, respectively. In a second run of the experiment, cucumber and muskmelon strains caused wilting on muskmelon at 7.0 and 6.9 dai, respectively, whereas summer squash seedlings wilted at 23.6, 29.0 and 9.0 dai when inoculated with muskmelon-, cucumber-, and squash-origin strains, respectively. Our results provide the first evidence of genetic diversity within E. tracheiphila and suggest that strain specificity is associated with plant host. This advance is a first step toward understanding the genetic and population structure of E. tracheiphila.

Dissoconiaceae associated with sooty blotch and flyspeck on fruits in China and the United States.

Zasmidium angulare, a novel species of Mycosphaerellaceae, and several novel taxa that reside in Dissoconiaceae, were identified from a collection of apples and Cucurbita maxima (cv. Blue Hubbard) from China and the USA that exhibited sooty blotch and flyspeck (SBFS) signs on their host substrata. Morphology on fruit surfaces and in culture, and phylogenetic analyses of the nuclear ribosomal DNAs 28S and internal transcribed spacer regions, as well as partial translation elongation factor 1-alpha gene sequences in some cases, were used to delineate seven previously unidentified species and three known species. Pseudoveronaea was established as a new genus of Dissoconiaceae, represented by two species, P. ellipsoidea and P. obclavata. Although Pseudoveronaea was morphologically similar to Veronaea, these fungi clustered with Dissoconiaceae (Capnodiales) rather than Chaetothyriales (Herpotrichiellaceae). Ramichloridium mali comb. nov., and three novel species, R. cucurbitae, R. luteum and R. punctatum were closely related with R. apiculatum, which together formed a distinct subclade in Dissoconiaceae. Species of Dissoconium s.lat. clustered in two well-supported clades supported by distinct morphological and cultural features. Subsequently Uwebraunia, a former synonym of Dissoconium, was resurrected for the one clade, with new combinations proposed for U. australiensis, U. commune, U. dekkeri and U. musae. Furthermore, we also reported that D. aciculare, Dissoconium sp., U. commune and U. dekkeri were associated with SBFS on apples.

Temporal patterns in appearance of sooty blotch and flyspeck fungi on apples.

Sooty blotch and flyspeck (SBFS) is a complex of about 80 fungal species that blemish the surface of apple fruit in humid regions worldwide. The dark colonies become visible in mid- to late summer, reducing the value of fresh fruit. Although many SBFS species can co-occur in the same orchard and even on the same apple, little is known about temporal patterns of these species, including the timing of colony appearance. To test the hypothesis that colonies of SBFS species appear on apples at characteristic times during the growing season, 50 apples were monitored weekly at three Iowa orchards in 2006 and six orchards in 2007 and 2008. However, a mean of 24.3 apples per orchard was assessed at harvest because of apple drop throughout the season. Colonies were marked with colored pens as they appeared. After harvest and after storage of apples at 2 °C for 3 months, SBFS colonies on each fruit were counted and classified by morphology, and a representative subset of colonies was excised from the fruit and preserved on dried peels for species identification using rDNA. Seventeen species were identified. Stomiopeltis spp. RS1 and RS2 appeared on apples 10 to 14 days before other SBFS taxa. Dissoconium aciculare was generally the last species to appear on apple fruit, and it continued to appear during postharvest storage. The most prevalent taxa in Iowa orchards were also the most abundant. Diversity of SBFS fungi in an orchard was positively correlated with cumulative hours of surface wetness hours due to rainfall or dew, which is believed to favor growth of SBFS fungi. Species-specific information about temporal patterns of appearance on apple fruit may lead to improved SBFS management strategies.

Epiphytic Survival of Erwinia tracheiphila on Muskmelon (Cucumis melo L.).

Erwinia tracheiphila, the causal agent of bacterial wilt of cucurbits, is transmitted by striped (Acalymma vittatum) and spotted (Diabrotica undecimpunctata howardi) cucumber beetles. Transmission occurs when infested frass with E. tracheiphila is deposited on plant surfaces with fresh feeding wounds. However, it is unclear whether the pathogen can survive as an epiphyte on leaves. Experiments were conducted in controlled environments to monitor E. tracheiphila survival on muskmelon (Cucumis melo) leaves under various temperature and moisture conditions. In the first experiment, muskmelon seedlings that had been spray inoculated with a rifampicin-resistant strain of E. tracheiphila were incubated at 10, 15, 20, 25, 30, or 35°C (±2°C) at ≥95% relative humidity, and E. tracheiphila populations were monitored for 72 h. In the second experiment, E. tracheiphila was monitored during alternating 12-h wet and dry periods, or continuous wet or dry conditions for 48 h at 20°C. Survival of E. tracheiphila on wet muskmelon leaves depended on temperature (P < 0.01), with the greatest survival at 10 and 15°C and least at 30 and 35°C. Leaf wetness also impacted survival; an initial 12-h dry period resulted in a 1,000- to 10,000-fold reduction in population size, followed by stabilization of the surviving population. These results demonstrate that E. tracheiphila can survive on muskmelon leaves under a wide range of environmental conditions, suggesting that epiphytic populations might serve as a reservoir of inoculum for infections.

Feasibility of Delaying Removal of Row Covers to Suppress Bacterial Wilt of Muskmelon (Cucumis melo).

Bacterial wilt, caused by Erwinia tracheiphila, is a major disease of cucurbit crops in the United States. Management of the disease relies on controlling two vector species, striped (Acalymma vittatum) and spotted (Diabrotica undecimpunctata) cucumber beetles. Six field trials were conducted at Iowa State University research farms during 2007, 2008, and 2009 to assess the efficacy of delayed removal of spunbond polypropylene row covers to control bacterial wilt on muskmelon (Cucumis melo). Treatments were (i) row cover removed at anthesis (conventional timing of removal), (ii) covers removed 10 days after row cover ends were opened at anthesis, (iii) covers removed 10 days after bumble bee hives were inserted under row covers at anthesis, and (iv) a noncovered control. In two field trials during 2007 and 2008, the delayed-removal row-cover treatments significantly suppressed bacterial wilt throughout the growing season and enhanced yield compared with the noncovered and removal-at-anthesis controls. In Gilbert in 2008, however, bacterial wilt suppression was equivalent among all three row-cover treatments. No bacterial wilt was observed during three trials in 2009, and there was minimal difference in marketable yield among treatments. Net returns were compared using partial budget and sensitivity analyses. Melon prices and occurrence of bacterial wilt had a strong impact on net returns. Using row covers increased production costs by 45%. In site years in which bacterial wilt occurred, delaying removal of row covers resulted in the highest returns. When bacterial wilt was absent, however, the delayed-removal row-cover treatments had the lowest returns. Results of the sensitivity analysis indicated that delaying removal of row covers for 10 days could be a cost-effective component of an integrated bacterial wilt suppression strategy for muskmelon where bacterial wilt occurs ≥50% of production seasons.

First Report of Microcyclosporella mali Causing Sooty Blotch and Flyspeck Disease on Plum in Poland.

Sooty blotch and flyspeck (SBFS), a disease caused by a complex of fungi, results in substantial economic losses for commercial growers of scab-resistant apple (Malus × domestica Borkh.) cultivars in Poland. However, many species causing SBFS in Poland are unidentified and sources of inoculum are uncertain. In August 2009, signs of SBFS were noted on fruit of plum (Prunus domestica L., cvs. Sweet Common Prune and Oullins Golden Gage) in orchards near Mostki in central Poland. Colonies consisted of olive green-to-black mycelial mats with few sclerotium-like bodies; infections ranged in severity from scattered spots to nearly complete coverage of the fruit surface. Ten of these colonies were isolated on potato dextrose agar (PDA). After 10 days of incubation at 22°C, total DNA was extracted; amplification of the internal transcribed spacer (ITS) region of rDNA utilized primers ITS1 and ITS4 (1). Nucleotide sequences were analyzed by ClustalW and compared with sequences in GenBank using BLAST. Sequences showed 99 to 100% homology to Microcyclosporella mali (2), which was formerly assigned as Pseudocercosporella sp. (1). Sequences from five isolates were submitted to GenBank (Accession Nos. HM101275, HM101276, HM101277, HM101278, and HM101279). Morphological characteristics-conidiogenous cells integrated, sympodial and polyblastic; conidial scars nonthickened and inconspicuous; conidia hyaline, subcylindric, narrow, straight or very slightly curved, truncate at the base and obtuse at the apex, often catenulate in simple or branched chains, with one (commonly) to five septa (12.5 × 2.6 to 50.7 × 4.0 µm)-were consistent with descriptions of M. mali (2). To fulfill Koch's postulates, each of the 10 isolates was used to inoculate three healthy apple fruit (cv. Golden Delicious) that had been previously washed under tap water and disinfested with 70% ethanol. After fruit were swabbed with cotton plugs that had been saturated with a suspension of spores in sterile distilled water (SDW), inoculated fruit were placed on filter paper that had been moistened with SDW, then sealed in foil bags and incubated at 22°C. When bags were removed 5 weeks later, dark colonies had appeared on the fruit. Isolates obtained from these colonies were morphologically identical to those used for inoculation. Control (SDW-inoculated and noninoculated) fruit that were incubated in the same manner developed no colonies. To our knowledge, this is the first report of SBFS on plum caused by M. mali in Poland; it had previously been noted as part of the SBFS complex on apple in Germany and Slovenia (2) and on apple and plum in the United States (3). References: (1) J. C. Batzer et al. Mycologia 97:1268, 2005. (2) J. Frank et al. Persoonia 24:93, 2010. (3) J. Latinovic et al. Plant Dis. 91:1685, 2007.

Novel fungal genera and species associated with the sooty blotch and flyspeck complex on apple in China and the USA.

Fungi in the sooty blotch and flyspeck (SBFS) complex cause blemishes on apple and pear fruit that result in economic losses for growers. The SBFS fungi colonise the epicuticular wax layer of pomaceous fruit but do not invade the cuticle. Fungi causing fuliginous and punctate mycelial types on apple are particularly difficult to identify based on morphological criteria because many species in the SBFS complex share the same mycelial phenotypes. We compared the morphology and nuclear ribosomal DNA phylogeny (ITS, LSU) of 11 fungal strains isolated from SBFS blemishes on apple obtained from two provinces in China and five states in the USA. Parsimony analysis, supported by cultural characteristics and morphology in vitro, provided support to delimit the isolates into three novel genera, representing five new species. Phaeothecoidiella, with two species, P. missouriensis and P. illinoisensis, is introduced as a new genus with pigmented endoconidia in the Dothideomycetes. Houjia (Capnodiales) is introduced for H. pomigena and H. yanglingensis. Although morphologically similar to Stanjehughesia (Chaetosphaeriaceae), Houjia is distinct in having solitary conidiogenous cells. Sporidesmajora (Capnodiales), based on S. pennsylvaniensis, is distinguished from Sporidesmium (Sordariomycetes) in having long, multiseptate conidiophores that frequently have a subconical, darkly pigmented apical cell, and very long, multi-euseptate conidia.

First Report of Sooty Blotch and Flyspeck Caused by Species of Dissoconium, Mycosphaerella, and Peltaster on Hawthorn Fruit in China.

Sooty blotch and flyspeck (SBFS), a disease complex comprised of as many as 30 putative species of fungi, occurs on the cuticle of pome fruits in moist production regions worldwide, inciting cosmetic damage that causes significant economic losses (1). Chinese hawthorn (Crataegus pinnatifida Bge.) is an economically important tree species in China. Its fruit are sold fresh or dried and are used as a culinary spice as well as an ingredient in Chinese traditional medicine. In October of 2007, Chinese hawthorn fruit exhibiting SBFS signs were sampled from supermarkets in Yangling, Shaanxi Province and Luoyang, Henan Province, China. Thalli directly from the hawthorn fruit were transferred onto potato dextrose agar (PDA) slants under a dissecting microscope and cultured at 22 ± 1°C in darkness. DNA was extracted from pure isolates and the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) was amplified and sequenced using primers ITS-1F and ITS4 (3). Phylogenetic analysis of the ITS sequences revealed that the 35 isolates generated in this study included five species in three genera: Dissoconium sp. (18 isolates), Mycosphaerella sp. (5 isolates), and Peltaster sp. 1 (4 isolates), Peltaster sp. 2 (4 isolates), and Peltaster sp. 3 (4 isolates). To fulfill Koch's postulates and verify that these fungi could also infest apple fruit, two representative isolates of each putative species were inoculated onto mature intact hawthorn and apple (cv. Fuji) fruit that had been surface disinfested with 75% ethanol and allowed to dry. Inoculum was prepared by comminuting 1-month-old cultures growing on PDA into a suspension of mycelial fragments and conidia (105 to ~106 CFU/ml) in a blender with sterile deionized water (SDW). Each isolate was inoculated on three hawthorn and three apple fruit by using cotton swabs. As controls, two surface-disinfested hawthorn and apple fruit were swabbed with SDW. After the inoculated hawthorn and apple fruit had been incubated in a moist chamber at 22 ± 1°C for 1 month, all inoculated hawthorn and apple fruit exhibited SBFS signs similar to those of the original colonies on hawthorn fruit, but the controls did not. Reservoir hosts have been inferred to play an important role in SBFS by providing the fungi with overwintering habitat and inoculum for infestations on apple. Many reservoir hosts have been reported in the United States and Japan (2). To our knowledge, this is the first report of fungi in the SBFS complex on hawthorn fruit and the first confirmation that fungi growing on hawthorn fruit can produce SBFS signs on apple fruit. These results identify hawthorn as a potential inoculum source for SBFS in apple orchards. References: (1) J. C. Batzer et al. Mycologia 97:1283, 2005. (2) K. Hemnani et al. Phytopathology 98(suppl):S66, 2008. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Development of a Nested Polymerase Chain Reaction Assay for Detection of Colletotrichum acutatum on Symptomless Strawberry Leaves.

A nested polymerase chain reaction (PCR) assay was developed for detection of Colletotrichum acutatum on symptomless strawberry leaves. In pure culture, the assay detected as little as 1.0 fg of DNA extracted from mycelium and as few as 1.5 conidia ml-1 when conidial suspensions were sonicated. On detached inoculated leaves, three alternative protocols to dislodge the pathogen were assessed: (i) immersion of whole leaves in 0.05% Tween 20 and manual agitation in plastic bags for 1 min (A); (ii) immersion in Tween 20, sonication for 30 min, then agitation for 1 min (SA); and (iii) freezing for 3 h, incubation for 2 days at 27°C, immersion in Tween 20, then sonication for 30 min and agitation for 1 min (FISA). Each method removed significantly (P ≤ 0.05) more conidia from leaves than the nontreated control; however, removal of appressoria did not vary among assays. In composite samples of noninoculated and inoculated (1.5 ×103 conidia ml-1) strawberry leaves, the nested PCR assay using the FISA protocol detected C. acutatum in as few as 1 infested leaf in 50 noninfested leaves. In a strawberry field, the assay detected the presence of C. acutatum in samples of asymptomatic strawberry leaves, showing potential as a powerful tool for reliable diagnosis of the pathogen in the field.

First Report of Seven Species of Sooty Blotch and Flyspeck Fungi on Asimina triloba in Iowa.

Fungi in the sooty blotch and flyspeck (SBFS) complex cause major economic losses on cultivated pome fruits in humid regions worldwide and also colonize many species of reservoir host plants. In 2007, 10 mature fruit of pawpaw (Asimina triloba), a native tree in eastern North America exhibiting SBFS colonies on the epicuticular wax layer, were collected from wild trees in eastern Iowa. Colonies of SBFS fungi on the fruit were described according to mycelial type (1). Isolates of representative colonies on acidified water agar were subcultured on potato dextrose agar and the morphological characters were observed. After DNA was extracted from cultures and amplified by PCR using primer set ITS-1F/ITS4, 470-bp sequences were compared with those of previously identified SBFS species using NCBI BLAST. The BLAST analysis showed 100% homology of the sequences with six species that had been previously confirmed to cause SBFS on apple fruit by fulfilling Koch's postulates (1): Colletogloeum sp. FG2, Dissoconium aciculare, Peltaster sp. P2.1, P. fructicola, Stomiopeltis versicola, and Stomiopeltis sp. RS1 (GenBank Accession Nos. AY598907, AY598874, AY5988888, AY598887, AY5160165, and AY598882, respectively). Using the NCBI bl2seq application, Dothideomycete sp. CS2, an additional previously confirmed SBFS species, was revealed by sequence homology. Morphology of some SBFS species on pawpaw differed from that on apple. For example, Colletogloeum sp. FG2, which produces the fuliginous mycelial type (1) on apple fruit, developed the ridged honeycomb mycelial type on pawpaw fruit. D. aciculare and Stomiopeltis sp. RS1 produced the compact speck mycelial type on pawpaw, but are known to develop discrete speck and ramose mycelial types, respectively, on apple. These differences may result from host species differences in the epicuticular wax layer of the fruit. To our knowledge, this is the first report of SBFS fungi on A. triloba in North America, although the SBFS species Zygophiala jamaicensis was reported on the same host in Japan (2). Identifying SBFS fungi on reservoir host plants is an important step toward improving disease management strategies. References: (1) J. Batzer et al. Mycologia 97:1268, 2005. (2) H. Nasu and H. Kunoh Plant Dis. 71:361, 1987.

First Report of Anthracnose Fruit Rot Caused by Colletotrichum acutatum on Strawberry in Korea.

Symptoms typical of anthracnose fruit rot; sunken, dark brown lesions on maturing fruits, were found in a commercial field of strawberry (Fragaria × ananassa) cv. Cal Giant in Yangyang County, Korea in May 2007. Masses of conidia were produced in acervuli in the center of lesions. The fungus was isolated on potato dextrose agar (PDA). Colonies grown on PDA were pale to mouse gray and became dark green to black in reverse. Conidia were formed in orange-to-salmon pink masses in the center of the culture. The average size of conidia on PDA was 15.2 × 4.6 µm, and they were hyaline, straight, cylindrical, with pointed ends, and aseptate (1). The fungus did not form an ascigerous stage in culture. Mycelial growth rate was 7.5 mm per day at 25°C on PDA. The identity of two isolates was confirmed as Colletotrichum acutatum J.H. Simmonds by PCR amplification using species-specific primers TBCA and TB5 (2), resulting in a characteristic 330-bp band on agarose gel. Morphological characters were in accordance with previous reports on C. acutatum. A pathogenicity test was conducted with five healthy plants of cvs. Cal Giant, Maehyang, Seolhyang, Kumhyang, Akihime, and Redpearl. After fruits and flowers were sprayed with a conidia suspension (105 conidia per ml), the plants were maintained at 10 to 25°C and 100% relative humidity in a greenhouse. As a control, five healthy plants were sprayed with sterile distilled water and incubated under the same conditions. Dark brown, water-soaked spots appeared on mature fruits of all cultivars after 5 days, and lesions on green fruits appeared on individual achenes. Flowers developed dark lesions, dried out, and died. No symptoms were found on the control plants. After the pathogen was reisolated from fruits and flowers lesions, the morphological characters developed in culture as described above. To our knowledge, this is the first report of C. acutatum causing strawberry anthracnose in Korea. References: (1) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990. (2) P. Talhinhas et al. Appl. Environ. Microbiol. 71:2987, 2005.

Adaptation of an Apple Sooty Blotch and Flyspeck Warning System for the Upper Midwest United States.

A warning system for sooty blotch and flyspeck (SBFS) of apple, developed in the southeastern United States, uses cumulative hours of leaf wetness duration (LWD) to predict the timing of the first appearance of signs. In the Upper Midwest United States, however, this warning system has resulted in sporadic disease control failures. The purpose of the present study was to determine whether the warning system's algorithm could be modified to provide more reliable assessment of SBFS risk. Hourly LWD, rainfall, relative humidity (RH), and temperature data were collected from orchards in Iowa, North Carolina, and Wisconsin in 2005 and 2006. Timing of the first appearance of SBFS signs was determined by weekly scouting. Preliminary analysis using scatterplots and boxplots suggested that cumulative hours of RH ≥ 97% could be a useful predictor of SBFS appearance. Receiver operating characteristic curve analysis was used to compare the predictive performance of cumulative LWD and cumulative hours of RH ≥ 97%. Cumulative hours of RH ≥ 97% was a more conservative and accurate predictor than cumulative LWD for 15 site years in the Upper Midwest, but not for four site years in North Carolina. Performance of the SBFS warning system in the Upper Midwest and climatically similar regions may be improved if cumulative hours of RH ≥ 97% were substituted for cumulative LWD to predict the first appearance of SBFS.

First Report of Bitter Rot Caused by Colletotrichum acutatum on Apple in China.

Bitter rot of apple caused by Colletotrichum gloeosporioides was first reported in China in 1985 (3). In China, apples are grown on approximately 2 million ha, and bitter rot occurs in almost all production areas, with crop damage ranging from 30 to 70%. During the summer of 2007, fungi were isolated from apple fruit exhibiting bitter rot symptoms in 12 and 9 orchards in Shaanxi and Henan provinces, respectively, in China. Symptoms included 2- to 3-cm-diameter, sunken, brown lesions on the fruit surface that contained black, pinhead-size fruiting structures producing orange conidial masses under high humidity, similar to that of C. gloeosporioides. On potato dextrose agar (PDA), colonies were white, pale gray, or pale orange when grown at 25°C. Conidia were 8 to 16 × 2.5 to 4 µm, fusiform, pointed at one or both ends, one celled, thin walled, aseptate, and hyaline. Appressoria were 6.5 to 11 × 4.5 to 7.5 µm, clavate to circular, and light to dark brown. These characteristics matched published descriptions of C. acutatum (2). To confirm pathogenicity, three mature, healthy apples (cv. Fuji) were surface disinfested with 70% ethanol and then wounded with a sterile needle. After being inoculated with a spore suspension (1 × 105 conidia/ml) prepared from a 2-week-old culture on PDA, these apples were sealed in a plastic bag and incubated at 25°C. Symptoms appeared 3 to 5 days after inoculation and began to enlarge 7 days later, forming lesions with fruiting structures. Under high humidity, cream-to-salmon pink spore masses were produced on lesions. As the lesions enlarged, the rot progressed to the core of the fruit in a V-shaped pattern. When the pathogen was reisolated from lesions of inoculated fruit onto PDA and incubated at 25°C, colony and conidial morphology were identical to those of the original isolates. Tests were performed three times with similar results. PCR with species-specific primer pair CaInt2/ITS4 (1) of genomic DNA from the isolates resulted in an amplification product of approximately 490 bp, which is specific for C. acutatum. The sequences exhibited 99% similarity with those of C. acutatum isolates AB273195 from GenBank. Approximately 20 of 103 symptomatic fruit from the field survey yielded fungal cultures whose morphology was consistent with that of C. acutatum, whereas the other cultures were C. gloeosporioides and Botryosphaeria dothidea. To our knowledge, this is the first report of bitter rot of apple caused by Colletotrichum acutatum in China. References: (1) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996. (2) B. C. Sutton. Page 523 in: The Coelomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1980. (3) X. M. Wang. M.S. thesis. (In Chinese). College of Northwest Agriculture, Shaanxi Province, China, 1985.