Diaporthe ambigua Associated with Post-Harvest Fruit Rot of Kiwifruit in Chile.

Kiwifruit is an important, expanding commercial crop in Chile. Several fungi have been reported to be associated with post-harvest rots of kiwifruit worldwide (2). During 2011 and 2012, kiwifruit produced in the VI and VII regions of Chile, showing symptoms of inner rot, were investigated with the aim of identifying the disease agent. The affected fruits had brown pubescent skin at the stem end that became soft and lighter in color than the adjacent firm healthy tissues. A watery exudate and white to pale grayish mycelial mats frequently developed at the stem end of the fruit, causing a water-drop stain down the sides on the dry brown healthy skin. The underlying tissue accessed by peeling off the skin was usually water soaked, soft, and lighter green than the healthy tissue. A fermented sour odor occurred frequently on severely decayed fruits. After incubation at 25°C over 7 days on potato dextrose agar (PDA), white to grayish, pale aerial mycelial mats were recovered from fragments of symptomatic kiwifruit superficially disinfected with 95% ethanol. After 2 weeks, black, spherical pycnidia developed, bearing hyaline, ellipsoidal, biguttulate conidia (5.4 to 12.6 × 2.1 to 4.7 µm). After 3 weeks, abundant perithecia embedded in a distinct, black, elevated stroma developed. Perithecia were black, globose, 200 to 500 µm in diameter with necks sinuous, filiform, 550 to 840 × 50 to 95 µm. Clavate, sessile asci, 30.9 to 50.2 × 6.6 to 12.5 µm contained ascospores biseriate, hyaline, smooth, fusoid-ellipsoid, widest just above the septum, tapering towards both ends, medianly septate, constricted at the septum at maturity, with 1 to 2 guttules per cell ascospores, 5.9 to 9.7 × 2.5 to 4.3 µm. All colonies obtained from kiwifruit displayed the same morphological traits and were consistent with those of a Diaporthe sp. (1). The internal transcribed spacer (ITS) region was sequenced using ITS1/ITS4 primers (4). Analysis of ITS region of kiwifruit isolates Damb_12 and Damb_16 (GenBank Accession Nos. KC294545 and KC294544, respectively) revealed 100% nucleotide identity to Diaporthe ambigua (HM575420, HM575419, DQ286274, DQ286270, and DQ286271). Six millimeter plugs from fungal colonies growing on PDA at 25°C for 7 days were used to inoculate 15 healthy untreated, ripe 'Hayward' kiwifruits. Control fruits were inoculated with agar plugs. Inoculated fruits were incubated at 25°C and 80% relative humidity. After 7 days, white to pale grayish mycelial mats developed only on the inoculated fruits, releasing a water drop stain. The underlying tissue was lighter green and water soaked. D. ambigua was reisolated only from the inoculated fruits. D. actinidiae has been previously reported on kiwifruit canes in Chile (3); however, to our knowledge, this is the first report on the occurrence of D. ambigua (Phomopsis ambigua) on kiwifruit in Chile. The fungal isolates (no. 1 to 21) have been deposited in the Laboratorio de Fitopatología Frutal y Molecular, Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas de la Universidad de Chile. References: (1) J. C. Jansen van Rensburg et al. Studies in Mycol. 55:65, 2006. (2) L. Luongo et al. J. Plant Pathol. 93:205, 2011. (3) A. Palma and E. Piontelli. E. Bol. Micol. 15:79, 2000. (4) White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Neofusicoccum australe (Botryosphaeria australis), as a Branch Dieback Pathogen of Avocado Trees in Chile.

Since 2007, Chilean avocado (Persea americana Mill.) orchards have been exposed to several abiotic stress conditions, namely frost damage and drought, due to three consecutive seasons of cold winters and shortage of irrigation water. At the same time, a severe disease resulting in tree dieback of cv. Hass, specifically, was observed in north-central Chile. Symptomatic trees exhibited abundant dead twigs in the tree canopy, and wilted leaves remained attached to the twigs in autumn. Closer inspection revealed reddish-brown necrotic lesions on the bark of the dead twigs, which girdled these symptomatic branches. When the bark was removed, the wood below appeared dark brown, in contrast to the yellowish-green coloring of healthy. The fungus was also consistently isolated from rotted fruit. A Neofusicoccum sp. with a yellow colony was consistently isolated from the necrotic lesions on PDA and incubated at room temperature for 3 days. Conidia produced in black pycnidia growing on 2% water agar with sterilized pine needles were smooth, unicellular, hyaline, and with granular contents. One or two septa developed at germination, but rarely before. The average length of the conidia was 27.0 ± 0.9 µm, with a length/width ratio of 3.9 ± 0.2 µm. Based on culture and conidial morphology, the isolates were putatively identified as Neofusicoccum luteum (1). DNA sequence analysis of the rDNA internal transcribed spacer (ITS) region was conducted for four representative isolates using primers ITS1 and ITS4 (4). The sequence analysis of ITS region of kiwifruit isolate H1M4 (Accession No. KC330230) reveled 100% nucleotide identity to N. australe (FJ157187 to FJ157192) (3). Pathogenicity tests were conducted with stem inoculations of 2-year-old cv. Hass plants grow in plastic containers in a sand/lime/peat mixture. For each inoculated plant (n = 8), a 7-mm-diameter agar plug from the margin of a 3-day culture was used as inoculum after wounding the stem to the depth to 7 mm with a cork borer. Negative control (n = 8) were wounded and then 'mock-inoculated' with sterile agar plugs. The inoculation sites were wrapped with Parafilm. All plants were kept in a greenhouse. After 5 months, all inoculated plants showed bark cankers and necrotic lesions beneath the bark, which were 5.2 cm long (n = 8). No symptoms developed on the control plants. N. australe was recovered from the margin of the necrosis lesion of every inoculated plant, thus fulfilling Koch's postulates and confirming its pathogenicity. Botryosphaeraceae spp. are the commonly reported to have ability to survive endophytically in their host, causing disease only when the host is exposed to a stress condition (2). To our knowledge, this is the first report of N. australe as a pathogen of avocado in Chile. The fungal isolates (PaHass No. 1 to 4) were deposited in the Laboratorio de Fitopatología Frutal y Molecular, Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas de la Universidad de Chile. References: (1) A. J. L. Phillips. http://www.crem.fct.unl.pt/botryosphaeria_site/ Accessed November 20, 2011. (2) B. Slippers and M. J. Wingfield. Fungal Biol. Rev. 21:90, 2007. (3) B. Slippers et al. Mycologia 96:1030, 2004. (4) White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Xiphinema rivesi from Chile Transmits Tomato ringspot virus to Cucumber.

The dagger nematode, (Xiphinema rivesi Dalmasso), a member of the X. americanum group, was first reported in 2002 in Chile (3). X. rivesi is a vector of at least four North American nepoviruses including Cherry rasp leaf virus (CRLV), Tobacco ringspot virus (TobRSV), Tomato ringspot virus (TomRSV), and Peach rosette mosaic virus (PRMV) (2). TomRSV, first reported in Chile in 1984, was associated with raspberry decline and lately with brownline disease in D'Agen prune trees (1), however none of the Xiphinema spp. found in Chile have been reported to transmit this nepovirus. Two virus isolates, TomRSV (prune brownline isolate PBL-08) and Grapevine fanleaf virus (GFLV) (Yellow mosaic isolate GFLV-012), from the virus collection of the Departamento de Sanidad Vegetal, Universidad de Chile were used in transmission tests with a population of X. rivesi found in Chile. X. rivesi is not known to transmit GFLV and this virus was included as a check. The nematodes were extracted from soil from a D'Agen prune orchard, and transmission tests were done in compliance with the criteria proposed by Trudgill et al. (4). Cucumis sativus cv. National Pickling were grown in a growth chamber at 25°C and used as acquisition hosts and transmission bait plants. Acquisition hosts were mechanically inoculated with GFLV or TomRSV, displaying systemic symptoms in 15 to 20 days. Noninoculated cucumber plants were included as controls. Virus infection was confirmed by double-antibody sandwich (DAS)-ELISA before the introduction of nematodes to the soil. After a 20-day acquisition feeding period, the nematodes were wet screened from the soil and added to the healthy bait plants and allowed a 20-day inoculation feeding period. X. rivesi transmitted TomRSV but not GFLV. TomRSV bait plants developed systemic symptoms 5 weeks after the nematodes were transferred. Transmission of TomRSV was confirmed by testing leaf and root sap of bait plants in a DAS-ELISA. High virus concentrations were detected in the roots and leaves of TomRSV symptomatic plants. Bait plants on which nematodes had been allowed to feed following virus acquisition from GFLV-infected or from virus-free control plants tested negative by ELISA. No symptoms appeared on bait plants used for GFLV transmission or the control bait plants. To our knowledge, this is the first report of transmission of TomRSV with a Xiphinema population from Chile and South America. References: (1) J. Auger. Acta Hortic. 235:197, 1988. (2) D. J. F. Brown et al. Phytopathology 84:646, 1994. (3) G. Leal et al. Fitopatología 37:75, 2002. (4) D. L. Trudgill et al. Rev. Nematol. 6:133, 1983.

First Report of Verticillium Wilt of Gold Kiwifruit, Actinidia chinensis Cv. Hort 16A, Caused by Verticillium albo-atrum in Chile.

Gold kiwifruit, Actinidia chinensis Planch cv. Hort 16A, was first planted in Chile in 2003 and vines started dying within 2 years. By the end of the 2007-2008 growing season, as much as 80% of the plants in several orchards had died. The disease was characterized by a conspicuous reddish brown discoloration of the xylem and the sudden wilting and dieback of plants any time during the growing season. In the spring, entire plants or parts of plants failed to break buds. In others, the buds broke, but juvenile leaf clusters then wilted and died. On severely affected plants, scion watershoots wilted and died. The disease was often accompanied by shallow cracking of the bark and slight sponginess of the underlying cortex. The disease was apparently most severe in sites that had been planted to Gold kiwifruit immediately after removal of apple, pear, citrus, or grape. Orchards planted following long-term maize, wheat, or grass culture were almost disease free. A fungus was consistently isolated from symptomatic vascular tissue disinfected in 1% sodium hypochlorite and plated on potato dextrose agar. Conidiogenous cells were arranged in verticels; conidia were hyaline, elliptical, single celled, and measured 3.5 to 8.5 × 1.8 to 4.3 µm (average 5.5 × 2.5 µm). Dark, resting mycelium developed after 1 to 2 weeks of incubation. On the basis of these morphological characteristics, the fungus was identified as Verticillium albo-atrum Reinke & Berthier. Identification was confirmed by sequencing part of the internal transcribed spacer (ITS) region with primers ITS1 and ITS4. The sequence of a representative isolate showed high homology (98% identity over a length of 494 bp) with a DNA fragment (NCBI Accession No. 108476) of V. albo-atrum from alfalfa. To complete pathogenicity tests, 20 healthy, 1-year-old Hort 16A kiwi vines grafted on Hayward kiwifruit (A. deliciosa Chevalier) seedlings were inoculated by injection of 20 µl of 106 conidia/ml into stems of the scion. Twenty control plants were injected with an equal volume of sterile distilled water. Plants were held in a controlled environment facility at 24°C with 16 h of light per day. Eight weeks after inoculation, typical wilting and dieback symptoms developed on 90% of the plants. Control plants injected with water remained healthy. Verticillium wilt has never been reported on kiwifruit (A. deliciosa) in Chile. V. albo-atrum has a rather narrow host range and is mainly reported as a pathogen on alfalfa, hop, soybean, tomato, and potato (1). To our knowledge, this is the first report of V. albo-atrum causing wilt and dieback on Gold kiwifruit (A. chinensis) cv. Hort 16A. The fungal isolates have been deposited in the Plant Pathology Laboratory of the Sanidad Vegetal Department of Agricultural Sciences Faculty of University of Chile under the name Actinidia chinensis/V. albo-atrum No. 1 to 8. Reference: (1) E. K. Ligoxigakis et al. Phytoparasitica 30:511, 2002.

First Report of Black Foot Disease of Grapevine Caused by Cylindrocarpon macrodidymum in Chile.

Black foot disease, caused by Cylindrocarpon macrodidymum Halleen, Schroers & Crous, is reported damaging table and wine grapes (Vitis vinifera L.) for the first time in Chile. During the summer of 2006, 2- to 5-year-old grapevines showed reduced vigor, shortened internodes, and drying and dying shoots along with abnormal development of roots with growth parallel to the soil surface, necrotic root crowns, and development of secondary roots. Internal necrosis extended from the bark to the pith in diseased parts of the plants. Other symptoms included black discoloration of the wood, gum inclusions in xylem vessels, black streaks in the vascular tissue, and reduction in root biomass, with sunken, necrotic root lesions. Eighteen Cylindrocarpon isolates were collected from roots, vascular elements, and pith tissue of grapevines cultivars (Flame Seedless, Red Globe, Thompson Seedless, Merlot, Carmenere, and Cabernet Sauvignon) from 12 locations in Chile. The isolates were identified on the basis of morphological features. All isolates produced micro- and macroconidia (one to three septa) and chlamydospores in short and intercalary chains (1,4), and by internal transcribed spacer (ITS1-5,8S-ITS4) rDNA and ß -tubulin (BT1, and BT2) partial sequences, identical to those of C. macrodidymum (isolate USS074, GenBank Accession No. AY 997558 and isolate USSO150, GenBank Accession No. AY 997598) (2). Phylogenetic analyses placed these isolates in a clade closely related, but clearly distinct from other clades, to C. destructans and C. liriodendri (2,3). Pathogenicity tests were completed by drench inoculation onto 50 6-month-old rooted cuttings of 'Red Globe' with 25 ml of conidia suspension (106 conidia ml-1) obtained from four isolates. Ten control cuttings of 'Red Globe' were inoculated with an equal volume of sterile distilled water. The plants were incubated for 4 months in a controlled environment facility at 24°C. All isolates tested were pathogenic. In addition, they caused significant root rot (t-test of disease incidence, P = 0.0048) and no significant level of variation was detected between different isolates. C. macrodidymum was reisolated from the region of brown streaking in all the inoculated cuttings and was not isolated from the water-treated controls. To our knowledge, this is the first report of C. macrodidymum causing black foot disease on grapevine in Chile. References: (1) C. D. Booth. Mycol. Pap. (CMI) 104:1, 1966. (2) F. Halleen et al. Stud. Mycol. 50:431, 2004. (3) F. R. Mantiri et al. Can. J. Bot. 79:334, 2001. (4) E. Petit and W. D. Gubler. Plant Dis. 89:1051, 2005.

First Report of Fenhexamid Resistant Isolates of Botrytis cinerea on Grapevine in Chile.

Botrytis cinerea Pers. (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is a haploid, filamentous ascomycete that causes gray mold on many economically important crops in temperate regions, especially grapevine. The management of gray mold on table grape in Chile involves cultural and chemical methods. Currently, protection programs are based on several fungicide families (dicarboximides, anilinopyrimidines, mixture of anilinopyrimidines and phenylpyrroles, and hydroxyanilides [fenhexamid]). During the last 25 years, B. cinerea developed resistance to virtually all specific fungicides used to control gray mold. Field resistance to benzimidazoles, phenylcarbamates, and dicarboximides was detected soon after their introduction. Recent studies using PCR-duplex and specific primers for the detection of transposable elements on Chilean B. cinerea isolates recovered from different table grape cultivars corroborated the presence of two sibling cryptic populations, transposa and vacuma (3). Some vacuma isolates have shown natural resistance to fenhexamide (HydR1) and it has been separated into two groups on a molecular basis using a marker gene (Bc-hch): Group I, fenhexamid-resistant vacuma isolates; Group II, vacuma and transposa isolates sensitive to this fungicide (HydS) (2). Group I and II isolates can not interbred (1,2). Other B. cinerea resistant phenotypes, HydR2 and HydR3, have been reported as belonging to Group II (1,4). Single-spore isolates of B. cinerea (472) were collected from different table grape cultivars from 13 locations in the Chilean Central Valley. The isolation was done during harvest time from rotting berries. Fenhexamid (Teldor; Bayer CropScience, Monheim, Germany) was diluted to 10 µg a.i./ml and added to the solid medium (10 g of glucose, 1.5 g of K2HPO4, 2 g of KH2PO4, 1 g of (NH4)2SO4, 0.5 g of MgSO4·H2O, 2 g of yeast extract, and 12.5 g of agar in 1 liter) to reach concentrations of 0, 0.025, 0.05, and 0.1 µg a.i./ml. A 5-mm mycelial plug from each isolate of B. cinerea was cut from the edge of 4-day-old colonies placed in the center of petri dishes with the described fungicide-amended medium and incubated at 20°C for 5 days. Two measurements, octogonal diameters, were taken from each of three replicates per treatment. Means were calculated and the diameter of the inoculated plug was subtracted from each mean. For each isolate, a linear regression of the percent inhibition of mycelial growth versus the Log10 transformation for each of the four concentrations of fenhexamid was obtained. The 50% effective concentration of fenhexamid (EC50) was calculated with the regression equation for each isolate. So, 95.3% of B. cinerea isolates were sensitive (EC50 under 0.083 µg/ml), 1.9% were less sensitive (EC50 between 0.084 and 0.1 µg/ml), and 2.8% (13 isolates) were resistant EC50 values ranging from 0.1 to 8.4 µg/ml. Through PCR-restriction fragment length polymorphism, according to the Bc-hch gene restriction pattern, all resistant isolates analyzed belong to Group II of B. cinerea (Bc-hch2) (2). To our knowledge, this is the first report of fenhexamid resistant isolates of B. cinerea on grapevine in Chile and South America. It would be necessary to study the population dynamics of these isolates, although failure of botrytis control in the field with this compound has not been reported. References: (1) C. Albertini et al. Mycol. Res. 106:1171, 2002. (2) E. Fournier et al. Mycologia 97:1251, 2005. (3) T. Giraud et al. Mol. Biol. Evol. 14:1177, 1997. (4) P. Leroux et al. Phytoma 599:31, 2006.

Black Dead Arm and Basal Canker of Vitis vinifera cv. Red Globe Caused by Botryosphaeria obtusa in Chile.

Several years ago, Vitis vinifera cv. Red Globe vines (over 6 years old), started to show disease symptoms approximately 10 weeks after bud break. Symptoms first appeared on the leaves at the base of the shoots and then spread to other leaves, continuing to appear throughout the growing season. Two forms of the disease (severe and mild) were observed, each case leading to premature leaf fall. The severe form was characterized by dieback of one or more shoots accompanied by leaf drop and shriveling and drying of fruit clusters. The mild form was characterized by wine-red spots on the leaf margins or the leaf blade, which coalesced to form large zones of necrosis between the veins and the margins of the leaf. Fruit clusters may wither. If the bark is scraped off, a brown streak, 1 to 2 cm wide, was often seen in the wood. The streaking began at the base of the affected shoot and extended upward to the ground level, eventually resulting in a basal canker. Botryosphaeria obtusa (Schwein.) Shoemaker (anamorph = Sphaeropsis malorum Berk.) was isolated from 86% of samples from vines that were 6 to 10 years old from 12 locations in IV, V, VI, and metropolitan regions of Chile. Isolations were made from brownstreaked wood. Isolates were identified on the basis of a previous description (1,2) and internal transcribed spacer (ITSI-5.8S-ITS2) rDNA sequences identical to those of B. obtusa (culture KJ9356, GenBank Accession No. AF027759). B. obtusa is established as one of the main fungi associated with black dead arm of grapevine (2,3). Pathogenicity tests were completed by inoculating approximately 20 µl of mycelial suspension via injection into the pith of 16 single-node, rooted cuttings of V. vinifera cv. Red Globe. Sixteen control cuttings were injected with an equal volume of sterile distilled water. Twenty weeks after inoculation, all B. obtusa-inoculated cuttings exhibited brown streaks in the wood extending 50 to 60 mm from the point of inoculation. The wood streaking observed in inoculated plants was identical to symptoms observed in naturally infected black dead arm vines in the vineyard. No symptoms were observed in the controls. B.obtusa was reisolated from the region of brown streaking in all the inoculated cuttings. B. obtusa was not isolated from the water-treated controls. To our knowledge, this is the first report of B. obtusa causing black dead arm and basal canker on Red Globe grapevine in Chile. The fungal isolates have been deposited in the plant pathology laboratory of the Sanidad Vegetal Department of Agronomy Faculty of the University of Chile under the name V. vinifera/B. obtusa from N 1 to 16. References: (1) G. Cristinzio. Inf. Fitopatol. 28:21, 1978. (2) P. Larignon and B. Dubos. Phytoma 538:26, 2001. (3) A. J. L. Phillips. J. Phytopathol. 146:327, 1998.

First Report of Phaeomoniella chlamydospora on Vitis vinifera and French American Hybrids in Chile.

Phaeomoniella chlamydospora (W. Gams, Crous. M.J. Wingfield & L. Mugnai) Crous & Gams (= Phaeoacremonium chlamydosporum) was isolated during the growing seasons of 2003-2004 from roots, trunks, and cordons of grapevines, including cvs. Cabernet Sauvignon, Merlot, Pinot noir, Thompson seedless, Ruby seedless and root stock 3309C, and Kober 5BB, from 10 locations in V, VI, VII, and metropolitan regions of Chile. P. chlamydospora was isolated from 82% of samples from vines 2 to 18 years old that showed decline symptoms in the field. Isolates were identified on the basis of a previous description (1) and internal transcribed spacer (ITS1-5.8S-ITS2) rDNA sequences identical to those of P. chlamydospora isolated from Vitis vinifera (culture CBS 22995, GenBank Accession No. AF 197973). P. chlamydospora is established as a member of the petri and esca disease complex and as a pathogen of grapevines (2,3). Pathogenicity tests were completed by injecting into the pith of 50 single-node, rooted cuttings of Pinot noir and 3309C, approximately 20 µl of a 106 conidia per ml suspension, obtained from four isolates from Chile and one from California. Ten control cuttings of Pinot noir and 3309C were injected with an equal volume of sterile distilled water. Twenty-four weeks after inoculations, all P. chlamydospora-inoculated cuttings exhibited dark streaking of the vascular tissue extending 40 to 45 mm from the point of inoculation. The vascular streaking observed in inoculated plants was identical to symptoms observed in declining vines in the vineyard. No symptoms were observed in the controls. P. chlamydospora was isolated from the region of vascular streaking in 85% of inoculated cuttings. P. chlamydospora was not isolated from the water-treated controls. The reisolated P. chlamydospora was verified with means of morphological characters and polymerase chain reaction amplification with the species-specific primers (3). P. chlamydospora is widespread and readily isolated from declining grapevines in Chile and other grape growing regions of the world. To our knowledge, this is the first report of P. chlamydospora from the cultivars cited above in Chile. References: (1) M. Groenewald et al. Mycol. Res. 105:651, 2001. (2) L. sparapano et al. Phytopathol. Mediterr. (Suppl.)40:376, 2001. (3) S. Tegli et al. Phytopathol. Mediterr. 39:134, 2000.