Nonwounded Fruits Research Articles

Genome Sequence Resource for Colletotrichum scovillei, the Cause of Anthracnose Disease of Chili.

Colletotrichum species cause anthracnose disease on the economically important spice crop chili. A total of 24 Colletotrichum species are known to infect chili and cause anthracnose. C. scovillei belongs to the C. acutatum species complex, and it shows greater aggressiveness than other species, particularly in the case of inoculation onto the nonwounded fruits of chili plants. The current work introduces an initial Illumina-Nanopore hybrid draft genome for C. scovillei TJNH1 together with the related annotations. Knowledge of this genome sequence provides an important reference genome of C. scovillei and will help further understand the pathogenic mechanism of C. scovillei to plant.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Cucumber Fruit Rot Caused by Fusarium incarnatum in Mexico.

Cucumber (Cucumis sativus L.) is an important vegetable crop for Mexico, which is the fifth highest producer of this crop worldwide. In November 2019, a fruit rot disease characterized by the presence of white mycelial growth at both ends of the fruit was observed with an incidence of approximately 30% in a greenhouse production area in Sinaloa State (geographical coordinates: 24°35'25'' N, 107°26'21'' O). Culture of small disinfected sections from cucumber lesion edges was carried out on PDA medium at 27°C for seven days. Colonies had profuse aerial mycelium that was initially white and became beige with age, and light orange sporodochia were eventually produced. Macroconidia had 3 to 5 septa and were slightly curved and tapered at the apex with a foot shape at one end and measured 25.4±2.5 × 2.8±0.3 µm. Microconidia were fusoid in form mostly with 2 to 4 septa and measured 15.7±2.04 × 2.4±0.25 μm (n= 100). The morphological characteristics of this fungal isolate were similar to those of Fusarium incarnatum (Leslie and Summerell, 2006). To confirm the species identification, the internal transcribed spacer (ITS) region, calmodulin (CAL), β-tubulin (B-tub) and translation elongation factor 1-α (TEF1-α) genes were amplified and sequenced from two representative isolates: FPM01 and FPM02. These sequences were submitted to GenBank with accession numbers MT387313 and MT387314 for the ITS region, MT410503 and MT410506 for CAL, MT410502 and MT410505 for B-tub and MT410504 and MT410507 for TEF1-α for the FPM01 and FPM02 isolates, respectively. BLASTn analysis of the sequences showed between 99.33 and 100% identity with F. incarnatum sequence accession numbers MH865893, LN901598, AB587036 and MK328877, which corresponded to strains CBS 130313, ITEM 6748, MAFF 236521 and FIAD-1, respectively. Further BLAST searches in the FUSARIUM-ID database indicated that these sequences had between 99.76 and 100% identity with sequences of the F. incarnatum-equiseti species complex (FIESC) (FD_01682, FD_01640 and FD_01643). To fulfill Koch's postulates, healthy cucumber fruits were disinfected with 1% NaOCl for one min and then washed with distilled water. Ten nonwounded cucumber fruits were spray inoculated with a conidial suspension (1 × 103 conidia/mL), and five fruits wounded with a sterilized needle were inoculated with mycelial discs (6 mm) at three sites each. Distilled water and PDA medium discs were used as controls. All fruits were placed into plastic bags with 80% relative humidity and incubated at 25°C for three days. At 24 hours after inoculation (hai), a soft rot symptom was observed at the fruit poles on non-wounded fruits and around the inoculated area of wounded fruits, and white mycelial growth was observed at 48 hai for both cases. All inoculated fruit showed similar symptoms to those observed initially in the field, and the control fruits remained healthy. The cultures obtained from tissues of inoculated fruits were similar to those from the initially obtained isolates, thus confirming pathogenicity. Recently, Fusarium incarnatum has been reported in China, causing fruit rot in muskmelon (Cao et al., 2019) and peach (Zhang et al., 2020) and crown rot and leaf spot in cucumber (Mao et al., 2020; Gao et al., 2020). To our knowledge, this is the first report of cucumber fruit rot caused by F. incarnatum in Mexico.

First Report of Colletotrichum aenigma Causing Walnut Anthracnose in China.

English/Persian walnut (Juglans regia L.) is grown as an economically valuable crop in temperate and subtropical regions. In August of 2018, serious fruit anthracnose, with brown to black circular or subcircular or irregular sunken lesions (Fig.1A), occurred on walnut trees ("Xiangling" and "lvling") in 33 ha., 23 ha. and 20 ha. orchards in Lincheng and Neiqiu county, in Xingtai, Hebei, China. Diseased fruits were observed on 41% (19,000 trees), 31% (13,300 trees) and 34% (11,400 trees) walnut trees. Diseased leaves, with circular or irregular brown to gray sunken lesions, were observed on 2% (19,000 trees), 2% (13,300 trees) and 1% (11,400 trees) walnut trees. From each orchard, 25 diseased fruits and leaves were collected, respectively. Twenty-one single spore isolates were obtained from fruits of three orchards and none from leaves as described by Cai et al. (2009). Six representative isolates 1811-1, 1811-4, 1811-7, 1811-8, 1811-11 and 1811-18, two from each orchard, were selected for further study. Colonies on PDA grew 11.8 mm d-1 at 25℃ under a 12/12 h light/dark cycle for 7 d. The upper side of colonies was milky (Fig.1 B), and reverse side was dark brown to brownish yellow. A few acervuli were observed on colonies. Conidiogenous cells were cylindrical to clavate, 10.6-29.7 × 3.1-5.3 μm (mean=21.3 × 4.0 μm, n=30) (Fig.1F). Setae were not observed. Conidia were smooth-walled, aseptate, straight or slightly distorted, cylindrical with one end slightly acute or broadly rounded ends, and 16.6-21.6 × 6.0-7.5 μm (mean=19.2 × 6.7 μm, n=30) (Fig.1 C). Appressoria were mostly irregular in outline, deeply lobed or lightly lobed, gray brown to dark brown, 8.3-16.6 × 7.1-14.5 μm (mean=12.5 × 9.7 μm, n=30) (Fig.1 D-E). Microscopic features were similar to the description of C. aenigma (Weir et al. 2012). To further identify isolates, the ribosomal internal transcribed spacers (ITS), β-tubulin 2 (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS) and chitin synthase (CHS-1) loci of representative isolates were amplified using ITS4/ITS5, Bt2a/Bt2b, CL1/CL2, GDF1/GDR1, GSF1/GSR1 and CHS-79F/CHS-345R primers (Prihastuti et al. 2009; Carbone & Kohn 1999). Sequences of representative isolate 1811-1 were submitted to GenBank (ITS: MN893316, TUB: MN893317, CAL: MN893312, GAPDH: MN893314, GS: MN893315, CHS-1: MN893313). Maximum likehood analysis of sequences of representative isolates and reference sequences of Colletotrichum spp. from GenBank revealed that six isolates clustered together with C. aenigma ex-type culture ICMP18608, and the bootstrap value was 100% (Fig.2). Pathogenicity tests were conducted on walnut fruit as described by Wang et al. (2017, 2018) and Cai et al. (2009). 10 wounded and 10 nonwounded fruits ("Xiangling", 35 mm diameter) were inoculated with isolates 1811-1, 1811-7 and 1811-11 conidial suspension (106 spore/mL) obtained from 10 d colonies grown on PDA at 25℃, respectively. 10 wounded and 10 nonwounded fruits were inoculated with sterile water. Inoculated and control fruits were incubated in containers at 25℃ in a 12/12 h light/dark cycle. After 10 days, necrotic lesions were observed in all inoculated fruits. The pathogen C. aenigma was reisolated from all inoculated fruits but not from control fruits. To our knowledge, this is the first report of C. aenigma causing walnut anthracnose in China. It is urgent to control walnut anthracnose caused by different species of Colletotrichum.

Colletotrichum acutatum complex causing anthracnose on peach in Brazil

Anthracnose, caused by Colletotrichum spp., has caused damage and great losses in peach orchards in two Brazilian states, Santa Catarina (SC) and Sao Paulo (SP). There is limited information on the characterization of Colletotrichum spp. from those locations. In this study we characterized Colletotrichum spp. isolated from peach fruit from SC and SP, and compared them to a reference isolate of Colletotrichum gloeosporioides, using molecular and traditional tools. Molecular analysis revealed that all isolates (7) belong to C. acutatum species complex (Cac) and three of them are C. nymphaeae. Most of the isolates showed distinct characteristics among them for most of the variables assessed using traditional methods. The optimum temperature for mycelial growth ranged from 24 to 26 °C, for conidial germination it ranged from 22.7 to 24.6 °C, and for germ tube length the temperature ranged from 21.3 to 27.3 °C. Disease severity caused by the Cac isolates was similar when compared wounded and nonwounded fruits. The PrpCnSP13–19 isolate was the most aggressive. More severe anthracnose symptoms were observed in fruits inoculated and maintained at simulated temperatures of Sao Paulo state when compared to fruits inoculated and maintained at simulated temperatures of the states of Parana, Santa Catarina and Rio Grande do Sul. Our results suggest that there are different strains of Cac, with different level of aggressiveness and optimum temperatures for disease development.

First Report of Fusarium incarnatum-equiseti Species Complex Causing Fruit Rot of Peach (Prunus persica) in China

Peach (Prunus persica [L.] Batsch; Rosaceae) is an economically important deciduous fruit. China is the largest producer of peaches, providing >50% of world production. Fruit rot is a devastating problem both pre- and postharvest. In May 2019, 882 infected peach fruits covered with white fluffy masses and brown necrotic lesions were observed among 4,500 fruits investigated in a commercial orchard in Jinshan, Shanghai, China. Disease incidence was ∼20%. Infected fruits were collected and kept in separate paper bags. Small sections (5 × 5 mm) were excised from the boundary of necrotic and healthy tissues, surface sterilized with 70% ethanol for 30 s and 5% NaOCl for 90 s, rinsed twice with sterilized distilled water (DW), and aseptically placed onto PDA. After 2 days of incubation at 26°C, sections from new colonies were transferred onto fresh PDA and purified by the single-spore method (Yang et al. 2008). Colonies on PDA had white aerial mycelia at first that turned off-white to light yellow after 5 days. No microconidia were observed. Seven morphologically similar isolates were obtained and representative isolate FQ01 analyzed. Macroconidia were five to seven septate with a tapered and elongated apical cell and prominent foot-shaped basal cells. Apical cells were conically elongated, and basal cells were prominently shaped, 20.1 to 47.2 × 2.5 to 2.9 μm. Macroconidia were produced from monophialides on branched conidiophores. Chlamydospores were abundant in clumps or chains, ellipsoidal to subglobose with thick and roughened walls. The isolated strain was identified morphologically as a member of the Fusarium incarnatum-equiseti species complex (FIESC) (Leslie and Summerell 2006). To confirm identity of FQ01, the internal transcribed spacer (ITS) region and the translation elongation factor 1-α gene (EF1-α) were amplified using primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. Total genomic DNA was extracted using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-tek) according to the manufacturer’s instructions. Resultant sequences of FQ01 were obtained and submitted to GenBank (MN133053 for ITS and MN133052 for EF1-α). BLASTn analyses showed that sequences shared 99.83% identity with a sequence of F. equiseti for ITS (MH879250.1) and 100% with a sequence of F. equiseti (MK692895.1) for EF1-α. Further BLAST search in the FUSARIUM-ID database indicated that the sequences shared 100% identity with a sequence of FIESC for ITS (FD_01695) and 98.58% with a sequence of FIESC for EF1-α (FD_01627) (Geiser et al. 2004). Pathogenicity tests were conducted on wounded and nonwounded peach fruits with mycelial plugs and conidial suspension, respectively. Ten healthy and surface-sterilized fruits (cv. I9) were wounded using a sterilized needle and inoculated with mycelial discs (6 mm). Ten surface-sterilized nonwounded peach fruits (cv. Qiumi) were sprayed with a 10⁵ conidia/ml suspension. For the control, wounded and nonwounded fruits were inoculated separately using agar plugs and DW, respectively All fruits were enclosed in plastic bags and incubated in a growth chamber at 26°C, 90% relative humidity, and 12 h/12 h light (10,000 lux)/dark. After 5 days, the inoculated sites on wounded fruits had brown and sunken lesions. On nonwounded fruits, brown rot and white fluffy mycelia were observed after 1 week. No symptoms were observed on control fruit. The fungus was reisolated from the lesions of the inoculated plants and verified morphologically. This is the first report of FIESC on peach fruits in China. Owing to the range of mycotoxins produced, members of the FIESC are not only saprophytes and opportunistic plant pathogens but also highly associated with human and animal disease (Leslie and Summerell 2006). The pathogen presents a severe threat to peach quality and production, so further research should be implemented to find effective management strategies to prevent and control it.

Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia

Anthracnose of chili (Capsicum spp.) causes major production losses throughout Asia where chili plants are grown. A total of 260 Colletotrichum isolates, associated with necrotic lesions of chili leaves and fruit were collected from chili producing areas of Indonesia, Malaysia, Sri Lanka, Thailand and Taiwan. Colletotrichum truncatum was the most commonly isolated species from infected chili fruit and was readily identified by its falcate spores and abundant setae in the necrotic lesions. The other isolates consisted of straight conidia (cylindrical and fusiform) which were difficult to differentiate to species based on morphological characters. Taxonomic analysis of these straight conidia isolates based on multi-gene phylogenetic analyses (ITS, gapdh, chs-1, act, tub2, his3, ApMat, gs) revealed a further seven known Colletotrichum species, C. endophyticum, C. fructicola, C. karsti, C. plurivorum, C. scovillei, C. siamense and C. tropicale. In addition, three novel species are also described as C. javanense, C. makassarense and C. tainanense, associated with anthracnose of chili fruit in West Java (Indonesia); Makassar, South Sulawesi (Indonesia); and Tainan (Taiwan), respectively. Colletotrichum siamense is reported for the first time causing anthracnose of Capsicum annuum in Indonesia and Sri Lanka. This is also the first report of C. fructicola causing anthracnose of chili in Taiwan and Thailand and C. plurivorum in Malaysia and Thailand. Of the species with straight conidia, C. scovillei (acutatum complex), was the most prevalent throughout the surveyed countries, except for Sri Lanka from where this species was not isolated. Colletotrichum siamense (gloeosporioides complex) was also common in Indonesia, Sri Lanka and Thailand. Pathogenicity tests on chili fruit showed that C. javanense and C. scovillei were highly aggressive, especially when inoculated on non-wounded fruit, compared to all other species. The existence of new, highly aggressive exotic species, such as C. javanense, poses a biosecurity risk to production in countries which do not have adequate quarantine regulations to restrict the entry of exotic pathogens.

Developing a methodology for identifying brown rot resistance in stone fruit

Assays were conducted to understand the effect of various factors involved with screening for resistance to Monilinia fructicola in stone fruit. First, the effect of maturity heterogeneity was determined in a set of fruit graded into three apparent maturity classes according to the IAD index for measurements taken with a DA-Meter. Second, different conidia concentrations, incubation times and M. fructicola strains were evaluated to optimize the methodology. Furthermore, the effects of fruit disinfection on M. fructicola, Penicillium expansum and Rhizopus stolonifer growth were studied. Finally, the developed methodology was applied to a set of commercial varieties of stone fruit to determine its suitability for identifying the level of susceptibility to brown rot. The results obtained indicated significant differences between wounded and non-wounded fruit, inoculum concentration and incubation time. The effect of strain aggressiveness was also confirmed, whereas, in general, no significant differences were observed among the fruit collected at the same harvest date. Our results also show that disinfection of fruit prior to inoculation had an effect on the infection process. In addition, the use of this methodology in commercial varieties of stone fruit allowed distinctions to be made among the levels of susceptibility to brown rot. Hence, the developed methodology could be applied to resistance screening in breeding programmes, as well as studying the genetic basis of brown rot resistance.

Brazilian isolates of Monilinia fructicola from peach do not present reduced sensitivity to iprodione

The dicarboximide fungicide iprodione has been used in Brazilian peach orchards for more than three decades to control brown rot and there are no studies on sensitivity to Monilinia fructicola in the country. In addition, isolates with reduced sensitivity to other important groups of fungicides have already been reported in Brazilian isolates. The sensitivity of M. fructicola to iprodione was evaluated using discriminatory doses and concentration resulting in 50% inhibition of mycelial growth (EC50) values. In total, 174 isolates collected from the main peach production areas were characterized for fungicide sensitivity. Disease control efficacy (DCE) was determined by comparing lesion inhibition on iprodione treated fruits with those without fungicide treatment. No resistant phenotypes were found in the M. fructicola population and 71.8% of the isolates were extremely sensitive to iprodione. The EC50 values of the isolates ranged from 0.13 to 1.01 μg/mL. DCE in wounded and non-wounded fruit was greater than 85% providing an efficient control in controlled conditions. These results suggest that iprodione could be integrated into disease management programs to prevent the establishment of resistant populations as already reported to other fungicides.

First Report of Diaporthe nobilis Causing Postharvest Rot of Blueberry in Shandong Province, China

Blueberry (Vaccinium L.) has become increasingly popular in China over the last several years owing to its high nutritional value. During June 2015, a postharvest fruit rot was observed on blueberry cultivar Duke cultivated in Qingdao, Shandong, China, and stored 7 days at 4°C after harvesting. Infected fruits showed soft and rotted lesions, covered with grayish or white mycelium under high humidity. To identify the causal agent of the disease, tissues were excised from surface-sterilized fruits and cultured on potato dextrose agar (PDA). Fungal colonies developed from all pieces of diseased tissues. Most isolates were identified morphologically and molecularly as Botrytis cinerea, Penicillium expansum, and Alternaria alternata, the important postharvest fungal pathogens of blueberry in China (Dai et al. 2016). Seven previously unidentified isolates were obtained, and all had white mycelia on PDA. After incubation for 3 weeks at 25°C on PDA, abundant pycnidia developed and produced hyaline, fusiform, biguttulate α-conidia and one-celled, hyaline, filiform β-conidia. The α-conidia dimensions were 5.9 to 8.7 × 2.0 to 3.4 μm (average 7.3 × 2.6 μm) and β-conidia were 16.5 to 27.8 × 0.9 to 1.9 μm (average 22.3 × 1.3 μm, n = 50). Morphological characteristics suggested the identity of the fungal isolates to be Diaporthe nobilis. To confirm identification, DNA was extracted from the mycelium of three representative isolates (SDB1, SDB2, and SDB3). The rDNA internal transcribed spacer (ITS), a partial sequence of β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), and calmodulin (CAL) were sequenced using primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. The consensus sequences (GenBank accession nos. MG557846 for ITS, MG557847 for TUB, MG557848 for TEF1, and MG557849 for CAL) showed highly similar (99, 100, 99, and 99%) with the sequences of ITS (KJ609006), TUB (KC344121), TEF1 (KC343874), and CAL (KC343395) from D. nobilis (Gomes et al. 2013). Pathogenicity tests were performed on 120 mature surface-disinfested blueberry (cv. Duke) fruits with a conidial suspension (10⁶ α-conidia/ml) of the three isolates. Sixty fruits were stab-inoculated to a depth of 3 mm using a sterile needle. For each isolate, 20 wounded and 20 unwounded fruits were inoculated with a 10-μl droplet. Forty fruits with or without wounding were treated with sterile water to create controls. Fruits were incubated in a chamber with a 12-h photoperiod at 25°C and 80% humidity. After 7 days, typical symptoms of postharvest rot with softened areas 3.7 to 6.4 mm in diameter were observed at wound sites. Experiments were conducted three times, and the pathogen was reisolated each time and confirmed as D. nobilis based on morphological and molecular analysis. Noninoculated and nonwounded fruits showed no symptoms. To our knowledge, this is the first report of D. nobilis causing blueberry postharvest rot in China. To prevent the spread of blueberry rot diseases, more care should be taken to avoid wounding during or after harvest.

FIRST REPORT OF ANTHRACNOSE ON CHERIMOYA CAUSED BY COLLETOTRICHUM TROPICALE IN CUBA

Cherimoya (Annona cherimola Mill.) fruits with preharvest anthracnose symptoms were collected in Province Artemisa, Cuba, in April 2015. Necrotic, sunken lesions with orange spore masses were observed on 71% of the fruits. Single spore isolations on potato dextrose agar (PDA) were initially white and became light gray with scant aerial mycelium and abundant orange conidiomata in concentric rings. Conidia were 12.5-17.5 × 5-6.5 µm, subcylindrical with rounded ends, aseptate and hyaline, morphological characters consistent with Colletotrichum tropicale (Rojas et al., 2010). To confirm pathogen identity, the ITS region, the Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) and a partial sequence of the glutamine synthetase gene (GS) were amplified and sequenced (European Nucleotide Archive Accession Nos. LT853592, LT853593 and LT853594, respectively). A phylogenetic analysis using Bayesian inference with published ApMat, GS, and ITS data for Colletotrichum species in the C. gloeosporioides complex (Jayawardena et al., 2016) placed our isolate INIFAT 4144 in the C. tropicale clade. For assessing pathogenicity, five detached cherimoya fruits were inoculated with a suspension of 105 spores/ml by depositing 30 µl droplets of the suspension on the surfaces of nonwounded and wounded fruits with a sterile needle. Control fruits received sterile water. Typical anthracnose symptoms were observed in all inoculated fruits after 3 to 5 days. Control fruits presented no symptoms. The pathogen was consistently re-isolated from the infected fruits. To our knowledge, this is the first report of C. tropicale in Cuba and the first report on A. cherimola.

Association of Botryosphaeria Panicle and Shoot Blight of Pistachio with Injuries of Fruit Caused by Hemiptera Insects and Birds.

Panicle and shoot blight of pistachio caused by species of Botryosphaeriaceae fungi remains one of the major diseases of pistachio in California, with the highest potential for crop destruction because buds and fruit clusters (panicles) are killed. Pistachio fruit can also be damaged by various insect pests. For instance, hemipteran insects are considered major pests of pistachio and can attack maturing fruit, causing necrotic areas on the fruit epicarp (epicarp lesion). Surveys in pistachio orchards showed a putative association of punctures of pistachio fruit with infection by Botryosphaeriaceae fungi, resulting in panicle and shoot blight. This study was undertaken to determine any association of Botryosphaeriaceae fungi with fruit bearing epicarp lesions and whether hemipteran insects can actively carry propagules of the pathogens to healthy fruit and cause disease. Three large insects of the order Hemiptera (Thyanta pallidovirens, Acrosternum hilare, and Leptoglossus clypealis) were used in pathogen-spread and infection experiments. The most frequently isolated species of the family Botryosphaeriaceae in pistachio, Neofusicoccum mediterraneum, was used in these transmission experiments. Significantly higher incidence of fruit infected by N. mediterraneum was recorded when pistachio clusters were sprayed with a spore suspension of N. mediterraneum and caged with the above insects than the level of infected fruit sprayed similarly with the pathogen but caged without any insects. Furthermore, increasing the number of insects caged with pistachio clusters resulted in an increase of insect feeding punctures and, subsequently, disease incidence. Similarly, increasing the number of wounds per fruit made with a needle, simulating injuries caused by the insect's stylet, resulted in significantly higher incidence of infected fruit than for wounded fruit with fewer injured sites. In addition, bird-injured fruit were also associated with higher incidence of panicle blight than fruit not injured by birds, especially in orchards with high disease incidence. In laboratory experiments, wounding or applying fruit sap to nonwounded fruit resulted in significantly higher incidence of infected fruit than in nonwounded fruit without sap. In field experiments, wounding of fruit before or after inoculation with N. mediterraneum resulted in higher disease incidence than nonwounded and inoculated fruit. Additionally, fruit sap enhanced initial mycelial growth of N. mediterraneum. Protecting fruit clusters from injuries by hemipteran insects or birds using mesh cages significantly reduced the incidence of panicle and shoot blight. The association of wounding by hemipteran insects and birds with high incidence of infected pistachio fruit may explain partially the broad distribution of Botryosphaeria panicle and shoot blight among pistachio orchards of the central San Joaquin Valley, even in areas where inoculum sources are not nearby.

FIRST REPORT OF Botrytis cinerea CAUSING POSTHARVEST GRAY MOLD OF TEJOCOTE (Crataegus mexicana) FRUIT IN MEXICO

In Mexico, tejocote (Crataegus spp.) is grown commercially on a total area of more than 900 ha. During November and December 2013, fruits of Crataegus mexicana var. chapeado showing a gray and firm rot were collected in commercial markets in Puebla. Small pieces of decayed fruits were surface-disinfected for 1 min in a 2% sodium hypochlorite solution, rinsed in sterile distilled water, and plated onto potato dextrose agar (PDA). A fungus was isolated whose colonies were initially whitish but turned gray with age. Black and irregular sclerotia were formed after 14 days of incubation at 20°C. Conidiophores were erect, subhyaline and dichotomously branched, and bore unicellular, ovoid-ellipsoid, subhyaline conidia measuring 7.7-12.7x6.1-9.5 μm. Based on morphology, the fungus was identified as Botrytis cinerea. Genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA was amplified using the universal primers ITS5 and ITS4 (White et al., 1990). PCR products were purified and sequenced. The resulting sequence of 496 bp was deposited in GenBank (accession No. KM594622). BLAST search showed 100% identity with B. cinerea sequences KF010847 and KJ476697. To confirm the pathogenicity of the fungus, 10 tejocote fruits were surface-disinfected with 80% ethanol. A conidial suspension (1x106 spores ml-1) was sprayed on the surface of non-wounded fruits. Control fruits were sprayed with sterile distilled water. Typical gray mold symptoms with gray sporulating lesions were observed only on inoculated fruits after eight days. Koch s postulates were fulfilled when the pathogen was re-isolated from the diseased fruits. To our knowledge, this is the first report of B. cinerea causing postharvest fruit rot on tejocote in Mexico and worldwide.

First Report of Anthracnose Caused by Colletotrichum theobromicola on Barbados Cherry (Malpighia emarginata) in Brazil.

Barbados cherry, also called acerola, is a fruit originated from tropical America that is well-known for its high content of vitamin C and nutritional value. Anthracnose is one of the most common diseases on Barbados cherry. In Brazil, this disease is associated with Colletotrichum gloeosporioides sensu lato (2). In 2012, necrotic and sunken spots were observed on Barbados cherry fruit (cv. Rubra) in Sao Paulo State, Brazil, from which a Colletotrichum species was isolated on potato dextrose agar (PDA). The isolate was grown on PDA at 25°C and 12-h photoperiod under fluorescent light. The colony was gray on the upper surface and the reverse part was dark gray. Conidia (n = 50) were cylindrical to subcylindrical, hyaline, and 12 to 15 (avg. 12.7) × 3.8 to 5.9 (avg. 4.3) μm. Conidia length/width ratio was 2 to 3.6. Pathogenicity was confirmed on Barbados cherry fruit. Inoculation was carried out by depositing 40-μl droplets of a conidial suspension (1 × 105 conidia ml-1) on fruit wounded with a sterilized needle and on non-wounded fruit. Fruit were incubated in a moist chamber at 25°C. First symptoms appeared 3 and 5 days after inoculation on wounded and non-wounded fruit, respectively. No symptoms were observed on control fruit inoculated with water. Six isolates recovered from inoculated fruit showed the same morphological characteristics of the previous isolate. The DNA of the fungus was extracted by a CTAB protocol (1) and the sequences of ITS, GAPDH, ACT, CHS-1, TUB, and CAL genes (4) were generated. Sequences were used in BLAST searches in GenBank and were 100% similar to C. theobromicola, except for GAPDH. The ITS (KC566724) and CAL (KC566437) sequences matched strain ICMP 17099 (JX010285 and JX009588, respectively) with 100% identity. The BTUB (KC566148), GAPDH (KC566578), ACT (KC566870), and CHS-1(KC566292) sequences matched with the strains ICMP 18649 (JX010447, 100% identity), ICMP 17099 (JX009957, 99% identity, 1 pb), ICMP 18567 (JX009457, 100% identity), and ICMP 18613 (JX009771, 100% identity), respectively. The sequences were also compared with authentic culture of C. gloeosporioides (IMI 356878) and the identities were: ITS 99% (JX010148), CAL 91% (JX009729), BTUB 90% (JX010445), GAPDH 83% (GU174561), ACT 93% (JX009494), and CHS-1 98% (JX009747). Based on the multi-gene sequencing, the isolate was identified as C. theobromicola. C. theobromicola was described in 2010 (3) and it is considered as a widely distributed species occurring on different hosts in tropical and subtropical regions (4). This report shows the necessity of the identification of Colletotrichum species from tropical fruits to elucidate the etiology of anthracnose diseases of which C. gloeosporioides sensu lato is considered to be the causal agent. To our knowledge, this is the first report of C. theobromicola on Barbados cherry.

First Report of Diaporthe novem Causing Postharvest Rot of Kiwifruit During Controlled Atmosphere Storage in Chile.

Chile is considered the third major exporter of kiwifruits (Actinidia deliciosa (A. Chev.) C. F. Liang & A. R. Ferguson) worldwide after Italy and New Zealand (1). The genus Diaporthe Nitschke (anamorph: genus Phomopsis) has been reported as causing postharvest rot in kiwifruit (4). During the current study, 1,400 fruits arbitrarily collected from seven controlled atmosphere (CA) rooms after 90 days of storage conditions (2% O2, 5% CO2) determined that 21.5% of the fruit were affected by decay and 0.86% developed symptoms different than those caused by Botrytis cinerea, the main postharvest pathogen associated to kiwifruit. Symptoms were soft rot with brown skin that started at the stem-end and in severe cases affected the entire fruit. Internally, affected fruit showed browning and watery tissues. Twelve affected fruits were surface disinfested (75% ethanol) and small pieces of internal rotten tissues were placed on acidified potato dextrose agar (APDA) for 7 days at 20°C. Twelve isolates were obtained, and four of them were identified morphologically and molecularly as Diaporthe ambigua, a species that has been previously described causing rot in stored kiwifruits in Chile (2). However, eight other flat, white to grayish colonies with sparse dirty-white aerial mycelium at the edge of the dish were obtained (3). Black pycnidia contained unicellular, hyaline, biguttulate, oval to cylindrical alpha conidia, with obtuse ends of (7.9) 6.7 (5.3) × (2.9) 2.5 (2.1) μm (n = 30). These isolates were tentatively identified as a Diaporthe sp. The species identification was determined by sequencing comparison of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA (GenBank Accession Nos. KJ210020 to 24, KJ210027, and KJ210033) and a portion of beta-tubulin (BT) (KJ210034 to 38, KJ210041, and KJ210047) using primers ITS4-ITS5 and Bt2a-Bt2b, respectively. BLAST analyses showed 99 to 100% identity with D. novem J.M. Santos, Vrandecic & A.J.L Phillips reference ex-type (KC343156 and KC344124 for ITS and BT, respectively) (3). Eighteen mature kiwifruits cv. Hayward were inoculated using a sterile cork borer on the surface of the fruit and placing 5-mm agar plugs with mycelial of D. novem (DN-1-KF). An equal number of fruits treated with sterile agar plugs were used as negative controls. After 30 days at 0°C under CA, all inoculated fruit showed rot symptoms with lesions 7.8 to 16.4 mm in diameter. The same D. novem isolate was inoculated with 30 μl of a conidial suspension (106 conidia/ml) on the surface of 18 ripe kiwifruits that were previously wounded and non-wounded as described above. An equal number of wounded and non-wounded fruits, treated with 30 μl sterile water, were used as negative controls. All inoculated wounded fruits developed rot symptoms with necrotic lesions of 14.1 to 20.2 mm of diameter after 14 days at 25°C. Inoculated non-wounded and negative control fruits remained symptomless. Koch's postulates were fulfilled by re-isolating D. novem only from the symptomatic fruits. To our knowledge, this is the first report of rot caused by D. novem on kiwifruit during cold storage in Chile and worldwide. Therefore, both Diaporthe species appears to be associated to Diaporthe rot of kiwifruit in Chile.

First Report of Colletotrichum nymphaeae Causing Apple Bitter Rot in Southern Brazil.

Bitter rot (BR) is a common disease on apples (Malus domestica Borkh.) worldwide. Fruit infection often occurs in warmer weather (25 to 32°C) where high temperatures and humidity strongly favor disease development. Three species causing BR have been reported: Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and its sexual stage Glomerella cingulata (Stoneman) Spaulding & Schrenk, C. acutatum J. H. Simmonds, and recently C. fragariae in Uruguay (1). In February 2012, typical BR symptoms were observed on apple fruits (cv. Gala) in two orchards located in Santa Catarina, Brazil. Initial symptoms were light brown, slightly sunken lesions that enlarged over time, becoming dark brown and forming sunken or saucer-shaped depressions that extended toward the fruit core in a V-shaped pattern. Acervuli were sparse and the conidia masses varied from dark gray to pale orange in color. To attempt to identify the causal organism, isolations were made from lesions on fully ripened fruits. Pure cultures were obtained following monosporic isolation and grown on PDA at 25°C with a 12-h photoperiod under fluorescent light for 7 to 15 days. The color of the upper surface of the colony varied from white to gray and was orange on the underside. One hundred conidia for each isolate were measured and each was one-celled, hyaline, fusiform, and with a length and width ranging between 8.0 and 25.9 μm (avg. 14.7), and between 2.4 and 9.9 μm (avg. 5.6), respectively. After germination, conidia formed oval appressoria between 5.1 and 9.9 μm (avg. 7.3) × 3.4 and 7.8 μm (avg. 5.2). These morphological characters are consistent with the description of C. nymphaeae (Pass.) Aa (2). To examine pathogenicity, eight 'Pink Lady' apples were inoculated with isolates MANE 25, 137, 143, and 144. A isolate treatments were arranged in a completely randomized design with three replications. Fruits were inoculated with two drops of 10 μl (1 × 106 conidia per ml) using wounded and non-wounded sterile fruit surfaces. Sterile distilled water drops served as controls. Fruit were incubated in a moist chamber at 25°C with a 12-h photoperiod for 10 days. Symptoms were observed at the inoculation site 3 to 4 days after inoculation (DAI) on wounded and 5 to 6 DAI on non-wounded fruits. All of the isolates produced symptoms identical to those observed in Santa Catarina, and each isolate was re-isolated from the apple lesions, confirming Koch's postulates. Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA using ITS1/IT4 primers and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using GDF/GDR primers. Isolate sequences had 100% homology to C. nymphaeae. The nucleotide sequences were deposited in GenBank (KC840351, KC840352, KC840353, KC840354, KC875404, KC875405, KC875406, and KC875407). Species identified as C. nymphaeae are considered as part of the C. acutatum species complex and have been reported to occur on water lilies (Nymphaea alba) (3) and causing bitter rot on apples in Korea (2,4). To our knowledge, this is the first report of C. nymphaeae causing bitter rot of apples in southern Brazil.

First Report of Phytophthora palmivora Causing Fruit Rot of Fig (Ficus carica L.) in China

Fresh fig (Ficus carica L.) has been grown on a large scale in Beijing, China, since 2011. In late July 2012, a rot disease occurred on immature fruit of fig after a heavy rain (average daily rainfall 170 mm) in Fangshan District, Beijing, which caused about 30% incidence of green fruit on trees. The symptom first appeared as a water-soaked lesion that was covered with a white, fluffy mass of mycelia, followed by a soft, mushy rot of infected area on the fruit. To isolate the causal agent, mycelia and sporangia from 10 symptomatic fruits were suspended in sterile water, spread on potato dextrose agar (PDA) plates, and incubated at 25°C for 18 h. The isolates from each diseased fruit showed the same colonial characteristics. A single sporangium was isolated under a dissecting microscope and transferred onto PDA to obtain a pure culture. On carrot agar, the colony was white and homogeneous with tidy edge, with a few aerial hyphae. Sporangia were obpyriform with obvious papillae and measured 54.7 to 63.8 (59.3) × 26.5 to 36.3 (30.7) μm. The chlamydospores produced in culture were spherical. The pathogen was identified as Phytophthora palmivora based on the morphological characteristics (3) and confirmed with ITS sequences by PCR amplification using rDNA universal primers ITS1 and ITS4. The resulting sequence (Accession No. KC131229) had a 99% identity to that of P. palmivora (JQ354937) isolated from Pachira aquatica. Koch's postulates were conducted by inoculating six surface-sterilized figs with a PDA plug from a 7-day-old culture, with six noninoculated (PDA plugs only) fruits serving as controls. The inoculated fruits were incubated at room temperature in a plastic box covered with film. Symptoms similar to those on the naturally infected fruits began on wounded fruits 48 h after inoculation and on non-wounded fruits 60 h after inoculation, while the six control fruits remained healthy. P. palmivora was reisolated from the symptomatic fruit tissue. P. palmivora is one of the most severe pathogens on edible figs, being reported by Japanese in 1941 (2). Fruit rot of fig caused by the pathogen was reported in Florida in 1984 (1). To our knowledge, this is the first report of P. palmivora leading to fruit rot on fig in China.

First Report of Pilidiella granati Causing Dieback and Fruit Rot of Pomegranate (Punica granatum) in Iran.

Iran is the largest producer of pomegranate (Punica granatum) in the world, with more than 60,000 ha currently in production. In the spring of 2011, a decline and dieback of young pomegranate trees (7 to 10 years old) were observed in the Kheir area of Fars Province. Dieback and twig blight developed toward the lower part of the stem, resulting in death of aerial tree parts and growing suckers from roots. Surface-disinfected tissues of diseased plants were plated on potato dextrose agar (PDA) and malt extract agar media. Isolates were separated into two groups that had either pale green or white aerial mycelia and sporulated after 5 to 7 days at 25°C. Pycnidia were globose and black with thin, membranous, pseudoparenchymatic walls, 80 to 140 μm in diameter. Conidia were hyaline, one-celled, elongate to fusiform, straight, and 11 to 17 × 4 to 6 μm (average 14 × 4.7 μm). Cardinal minimum growth temperatures were 8 to 10°C, optimum at 27 to 30°C, and maximum at 35°C. Radial growth rate at 30°C was 8 to 9 mm per day. Representative isolates were deposited in the CBS-KNAW Fungal Biodiversity Centre, the Netherlands (CPC 19625 = CBS 130974 and CPC 19626 = CBS 130975; GenBank JN815312 and JN815313, respectively). Genomic DNA was extracted with the UltraClean Microbial DNA Isolation Kit (MoBio Laboratories, Inc., Solana Beach, CA) and the internal transcribed spacer (ITS) region of the nrDNA operon of two isolates were sequenced as described previously (1). On the basis of morphology (3), the causal organism was identified as Pilidiella granati Sacc. This identification was corroborated by the ITS sequence data, which was identical for both colony types to GenBank HQ166057 (identities = 614 of 614 [100%]). Pathogenicity tests were conducted using two representative isolates from each group on 5-month-old P. granatum trees with 10 replicates under greenhouse conditions; 5-mm mycelial plugs from the edge of 7-day-old colonies on PDA were placed under the bark of twig wounds. Uncolonized PDA plugs were used as noninoculated controls. Pathogenicity was also tested on nonwounded fruit by placing colonized 5-mm-diameter mycelial plugs on surface-disinfected pomegranate fruits; noncolonized PDA plugs were used as controls. All treated fruit were placed in plastic bags and maintained at 25°C for 10 days. Isolates were found to be pathogenic on twigs after 2 months, giving rise to brown lesions that were 2 to 5 cm long. No lesions were observed on the controls. Furthermore, the fungus was reisolated from all infected tissues, satisfying Koch's postulates. On pomegranate fruit, the fungus colonized the fruit after 5 to 8 days, followed by the appearance of fruit rot symptoms leading to the formation of abundant pycnidia covering the skin after 10 days. No decay was observed in control inoculations. Pilidiella granati has previously been reported as a pathogen of P. granatum fruit from Europe, Asia, and the United States (2). To our knowledge, this is the first report of this pathogen causing dieback and fruit rot of pomegranate in Iran.

First Report of Association of Mucor circinelloides on Noni (Morinda citrifolia) in Hawaii.

Noni (Morinda citrifolia) is a popular medicinal plant found in tropical or subtropical regions of the world. The fruit and juice extracts have properties that are reportedly therapeutic for diabetes, high blood pressure, and certain types of cancer (1,4). In our studies on noni juice produced from fruit collected from the Kohala and Puna districts of the island of Hawaii from 2008 to 2010, Mucor circinelloides f. sp. circinelloides was isolated from 85% of 157 juice samples and observed with up to 75% incidence on fruit surfaces during fermentation processing in glass jars. Fungal growth, appearing 14 to 21 days in storage at 22°C, was pale yellow to tan brown and was associated with wounded surfaces. Single-spore strains, KN 06-2 (2006; ripe fruit puree) and KN 08-08 (2008; fermented juice; CBS 124110), identified by Centraalbureau voor Schimmelcultures by molecular methods were 97.3% similar in internal transcribed spacer sequence to the type strain (CBS 195.68). M. circinelloides f. sp. circinelloides strains (KN 08-08, KN 09-06, or KN 10-02) (2008 to 2010; fermented juice) were inoculated by pipetting an aliquot of 100 μl of fungus strain spore suspension (1 × 105 to 1.33 × 106 spores/ml) onto firm, yellow maturity noni fruit that were washed, surface disinfected, and either wounded (surface cuts) or nonwounded. Controls consisted of no inoculation and sterile distilled water (SDW) inoculation treatments. Ten to twenty each of wounded and nonwounded fruit comprised each inoculation treatment. Fruit were incubated in acrylic bins with a layer of distilled water at the bottom, and sealed with snap-on lids. The bins were incubated on a lab bench at 22 to 23°C under fluorescent lights. Fruits were evaluated for presence of fungal growth and severity of symptoms. To determine viability of spores on inoculated fruit without symptoms, surfaces were swabbed with sterile cotton swabs dipped in SDW, streaked on potato dextrose agar (PDA) plates, and incubated at 22°C under fluorescent lights. The inoculation experiment was conducted twice. Nonwounded fruit inoculated with M. circinelloides f. sp. circinelloides strains did not result in infections (KN 09-06 and KN 10-02) or produced slight mycelial growth (0 to 20%; KN 08-08). Wounded fruit inoculated with any of the three strains resulted in 85 to 100% infection of moderate severity. There were no infections in noninoculated or SDW treatments of nonwounded or wounded fruit. Koch's postulates were fulfilled with the reisolation of M. circinelloides f. sp. circinelloides from selected fruit exhibiting soft tissue, discoloration, and sporulating yellowish green mycelial growth. Swab washes from asymptomatic surfaces of inoculated nonwounded fruit resulted in the growth of M. circinelloides f. sp. circinelloides on PDA, proving viability of the spores and confirmed that the fungus is primarily pathogenic only on wounded fruit surfaces. To our knowledge, this is the first report of M. circinelloides as a wound pathogen of noni fruit. The quality of fermented noni juice may be affected by the presence of M. circinelloides f. sp. circinelloides but can be remedied by pasteurization that does not affect antitumor properties (unpublished data). This fungus is also a reported pathogen of mango (2) and peach (3).

First Report of Fusarium solani Causing Fruit Rot of Sweet Pepper in Trinidad.

In Trinidad, sweet pepper (Capsicum annuum L.) is an important crop that is produced for local markets and regional export. From February to April 2010, severe fruit rot was observed in 9 of 11 commercial fields located in North Trinidad in the major production areas of North and South Aranguez. All fields were in the late harvesting stage and the most commonly grown cultivars were Aristotle and Canape. Disease incidence for each field was estimated to be 80% with a yield loss of 40 to 60%. Symptoms appeared on mature red fruits but growers reported that disease can also occur on green fruit. Symptoms began as soft lesions that turned dark brown to black. Lesions usually originated at the calyx end of the fruit and extended down the sides. Fruits were surface sterilized by rinsing with 70% ethanol for 2 min, followed by three rinses with sterile distilled water. Two 4-mm3 blocks of tissue from the opposite sides of fruit lesions were transferred to water agar and incubated for 5 to 7 days at 25 ± 1°C. A 4-mm3 agar block consisting of the leading mycelial edge was then transferred to potato dextrose agar (PDA) and incubated under the same conditions. Colonies on PDA were fast growing with white, fluffy, aerial mycelia; hyphae were septate and hyaline; conidiophores were unbranched; microconidia were abundant, thin walled, hyaline, ovoid, one to two celled, and measured 6 to 10 × 2 to 4 μm. Macroconidia were hyaline, three to four celled, curved, thick walled, and measured 20 to 30 × 4 to 6 μm. PCR amplification was carried out utilizing universal primers ITS4/5 and translation elongation factor primers EF1/2 (2). Sequence comparisons of the internal transcribed spacer (ITS) region (HM157262) and EF-1α gene (HQ014854) with cognate sequences available in GenBank and the FUSARIUM-ID databases revealed 100 and 99.6% sequence identity, respectively, to Fusarium solani (Mart.) Sacc. Pathogenicity tests were conducted by drop inoculating 10-μl of spore suspension (106 spores/ml) of each of four isolates on wounded and unwounded sites of mature sweet pepper fruits (five per isolate of cvs. Aristotle, Canape, Century, Destra, and Paladin). Control fruits were inoculated with sterile distilled water. Inoculated fruits were kept at 25 ± 1°C in loosely sealed plastic containers and monitored for the onset of symptoms for 6 days. The experiment was conducted twice. Lesions (8.0 to 15.2 mm in diameter) developed on wounded fruit of Aristotle, Canape, and Century. No symptoms were seen on Destra, Paladin, or the water controls. No symptoms developed on nonwounded fruits. Koch's postulates were fulfilled by reisolating the pathogen from infected tissues. Fruit rot caused by F. solani has been reported to be a serious constraint to sweet pepper production in Canada (4), the United Kingdom (1), and New Zealand (3). To our knowledge, this is the first report of Fusarium fruit rot of sweet pepper in Trinidad.

Gray Mold Infection of Actinidia arguta in Italy.

Gray mold is caused by Botrytis cinerea Pers.:Fr., a cosmopolitan and polyphagous fungus that is responsible for significant losses on several fruit crops, including kiwifruit (Actinidia deliciosa), in the field and during storage (2). A. arguta, the hardy kiwifruit, is grown in several countries (e.g., Japan, China, Korea, Russia, and the United States) (1,3) and regions of Italy (Apulia [southeastern], Basilicata [southern], Marche [central eastern], Tuscany [central western], and Piedmont, Friuli Venezia Giulia, and Trentino Alto Adige [northern]) (4). The oblong- to oval-shaped fruit is produced in clusters, has a smooth, edible, thin skin, bears tips with a persistent style, and weighs approximately 5 to 15 g (1,3,4). Several fruits of cv. Ananasnaya selection 'Anna red' harvested from September to October in 2007 and 2008 in the Apulia and Marche regions showed browning and depressions in the surface and a decay of the flesh. Disease affected approximately 5% of 420 examined fruits; symptoms mainly developed in the equatorial zone, eventually expanding through the entire fruit. Severely infected fruit showed deformation followed by drying. In the field or after storage in an environment with high relative humidity, fungal mycelia with sporulation appeared on the surface of the symptomatic areas of the fruit. Nineteen symptomatic fruit were surface disinfested by immersion in a 0.5% sodium hypochlorite solution for 2 min, then rinsed in sterile distilled water. Portions of the flesh were plated onto petri dishes containing potato dextrose agar (PDA) supplemented with 250 mg/liter of each ampicillin and streptomycin sulfate. Colonies that originated from symptomatic portions of the fruit were transferred to new PDA plates and identified as B. cinerea by the morphological features of conidiophores and conidia. Conidia were produced on dichotomously branched conidiophores, which had globose basal cells from mycelia. The conidia were hyaline or pigmented, ellipsoid-obovoid, globoid, and without septa (2). Conidia were collected from an isolate (Accession No. CBS 125087) of B. cinerea recovered from diseased A. arguta fruit grown in Monopoli (BA), Apulia in September 2007, and maintained in pure culture on PDA. A spore suspension was created by flooding plates with a small volume of sterile distilled water plus surfactant (0.05% Triton X-100). The suspension was filtered through four layers of cheesecloth and diluted with sterile water to an absorbance of 0.25 at 425 nm as determined by a spectrophotometer. This suspension contained approximately 1.0 × 106 conidia/ml and was diluted with sterile water to 1 × 104 spores/ml. Twenty microliter droplets of spore suspension were deposited within the equatorial zone on each of 10 nonwounded fruits of cv. Ananasnaya selection 'Anna red'. All fruit developed typical gray mold symptoms after 4 to 5 days of incubation at 20 ± 2°C and 95 to 98% relative humidity. Reisolation from the decayed tissues on PDA produced pure colonies of B. cinerea. To our knowledge, this is the first report of B. cinerea infection on A. arguta in Italy.