First Report of Stem Blight of Blueberry in California Caused by Neofusicoccum parvum

In July 2013 in coastal (Santa Barbara County) California, commercial plantings of southern highbush blueberry (Vaccinium corymbosum) developed symptoms of a previously undiagnosed disease. Symptoms consisted of reddening and wilting of foliage, with leaves and small twigs later drying up. The bark of diseased branches was discolored and sunken; removal of this bark revealed a brown discoloration of the underlying wood. Approximately 5% of the planting was affected. When placed on acidified potato dextrose agar (A-PDA), surface disinfested pieces of symptomatic wood consistently yielded one type of fungus. On A-PDA, isolates produced extensive white aerial mycelium that turned dark gray after 4 to 5 days and formed pycnidia after 21 days. Three single-spore isolates were grown on PDA for 21 days for morphological and molecular characterization. Conidia were hyaline, smooth, and ellipsoid with round apices and truncated bases. Conidia measured 13 to 20 × 5 to 7.5 μm (n = 50; mean 16.7 × 6.1 μm), with a length/width ratio of 2.73. After 25 days, conidia became biseptate with a darker middle cell. rDNA sequences of the internal transcribed spacer (ITS) region of the isolates (GenBank KJ126847 to 49), amplified using primers ITS1 and ITS4 (5), were 99% identical to the holotype isolate of Neofusicoccum parvum Pennycook and Samuels (3) by a BLAST query (GU251125). Partial sequences of the translation elongation factor 1-alpha (EF1-α) gene (KJ126850 to 52), obtained using primers EF728Fa and EF986R (5), were 99% identical to N. parvum (GU251257). To demonstrate Koch's postulates, 14-day-old colonies of the three N. parvum isolates were grown on A-PDA. Using three blueberry cultivars (Abundance, Jewel, and Snowchaser), slits were cut beneath the epidermis of branches 1 cm diameter or less; one colonized agar plug (6 mm diameter) was placed into each cut and the epidermis was resealed with Parafilm. Ten inoculations (one inoculation per branch; two branches per plant) were made for each isolate and each cultivar; inoculated plants were maintained in a greenhouse. After 10 to 14 days, leaves on inoculated branches turned red and wilted, bark above and below the inoculation sites turned brown, and vascular tissue beneath the bark was also brown. After 21 days, diseased areas became sunken. N. parvum was recovered from all inoculated branches of all cultivars and matched the characteristics of the original isolates. Control branches, inoculated with sterile agar plugs, did not develop any symptoms and N. parvum was not isolated. This experiment was repeated with similar results. Many Botryosphaeriaceae species, including N. parvum, are associated with canker and dieback symptoms on blueberry worldwide (2). To our knowledge, this is the first documentation of stem blight caused by N. parvum on blueberry in CA. Blueberry is a rapidly expanding industry in the state, with 960 ha planted in 2005 increasing to 2,830 ha in 2012 (1). Drought stress predisposes plants to stem blight caused by Botryosphaeriacease species (4); therefore, expansion into arid areas of CA could increase the incidence and severity of N. parvum. References: (1) N. Amer. Blueberry Council. 2012 World Blueberry Acreage & Prod. Rept., 2013. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., online publication, ARS, USDA. Retrieved February 5, 2014. (3) S. R Pennycook and G. J. Samuels. Mycotaxon 24:445, 1985. (4) W. A. Sinclair and H. H. Lyon. Diseases of Trees and Shrubs, Second Edition. Comstock Publ. Assoc. 2005. (5) B. Slippers et al. Mycologia 96:83, 2004.

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First Report of Fusarium oxysporum f. sp. lactucae Race 1 Causing Fusarium Wilt of Lettuce in Norway

Plant Disease ◽

10.1094/pdis-01-21-0134-pdn ◽

2021 ◽

Author(s):

Maria Luz Herrero ◽

Nina Elisabeth Nagy ◽

Halvor Solheim

Keyword(s):

Costa Rica ◽

Fusarium Oxysporum ◽

Vascular Tissue ◽

Uv Light ◽

Elongation Factor ◽

Aerial Mycelium ◽

Light Cycle ◽

Single Spore ◽

Fungal Isolates ◽

Race 1

Lettuce (Lactuca sativa L.) is produced in Norway both in field and greenhouses. In Norway, greenhouse lettuce is one of the most important vegetables grown year-round. In winter 2018, wilting symptoms were observed on soil-grown lettuce of the cultivar Frillice in a greenhouse in south east Norway (Buskerud county). Affected plants showed stunted growth, wilting of outer leaves, and brownish discoloration of vascular tissues of taproots and crowns. According to the producer, the disease led to an estimated 10% of yield losses. Fungal isolates were obtained from crowns and roots of diseased plants collected from the greenhouse in 2018 and 2019. Two single spore isolates, 231274 from 2018 and 231725 from 2019, were used in further studies. The isolates were incubated on synthetic nutrient-poor agar (SNA) at 18-20 ⁰C, and a 12 hours dark, 12 hours UV light cycle. Isolate 231274 produced abundant macro- and microconidia characteristics of Fusarium oxysporum while macroconidia were never observed in isolate 231725. On potato dextrose agar (PDA), colonies of isolate 231274 were purple in color and colonies of isolate 231725 were pinkish with abundant aerial mycelium. For PCR-assay, DNA from mycelia was extracted using Easy-DNA kit (Invitrogen). A portion of the translation elongation factor 1-α (EF1-α) gene was amplified using primers F-728F (Carbone and Kohn. 1999) and EF2 (O'Donnell et al. 1998) as described by Aas et al. 2018. Blast analysis of both sequences (accession no. MW316853 for 231274 and MW316854 for 231275) obtained a 99% homology with the sequence of Fusarium oxysporum f.sp. lactucae (FOL) race 1 strain S1 (accession no. DQ837657)(Mbofung et al. 2007). Both isolates were identified as race 1 by using specific primers Hani3’ and Hanilatt3rev (Pasquali et al. 2007) as described by Cabral et al. 2014. To complete Koch’s postulate, lettuce plants of the cultivar Frillice were used. Race identity was confirmed using the differential lettuce cultivars Costa Rica No.4 (resistant to FOL race 1), Banchu Red Fire (resistant to FOL races 2 and 4) and Romana Romabella (resistant to FOL races 1 and 2) (Gilardi et al. 2017) provided by the breeding company Rijk Zwaan (De Lier, The Netherlands). For inoculation, roots of six 2-weeks old seedlings per cultivar were dipped in a spore suspension (1 x 106 CFU/ml) for 1 min, while controls were dipped in distilled water. Seedlings were planted in 250 ml pots containing fertilized potting substrate, and were placed in a greenhouse with temperature ranging from 15 to 35 ⁰C and an average of 23 ⁰C. After 10 days reduced growth was observed in cultivars Frillice and Banchu Red Fire for both fungal isolates. After 25 days wilting was observed in both cultivars. Affected plants presented discoloration of vascular tissue. No difference in growth was observed between cultivars Romana Romabella and Costa Rica No. 4 and their respective controls. FOL was re-isolated from all inoculated cultivars but not from controls. The colony patterns of the recovered isolates were the same than those of the isolates used for inoculation. These results confirm that the isolate belongs to race 1. Greenhouse lettuce in Norway is mainly produced in hydroponics. FOL is here reported to cause damages in soil- grown lettuce. Nevertheless FOL in hydroponic systems has been reported in Japan (Fujinaga et al. 2003) and Thailand (Thongkamngam and Jaenaksorn 2017). Thus, the possibility of infections in hydroponics remain a big concern for lettuce production in Norway.

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First Report of Cankers and Dieback Caused by Neofusicoccum mediterraneum and Diplodia corticola on Grapevine in Spain

Plant Disease ◽

10.1094/pdis-05-11-0429 ◽

2011 ◽

Vol 95 (10) ◽

pp. 1315-1315 ◽

Cited By ~ 7

Author(s):

C. Pintos Varela ◽

V. Redondo Fernández ◽

O. Aguín Casal ◽

J. P. Mansilla Vázquez

Keyword(s):

Average Length ◽

Aerial Mycelium ◽

Its Region ◽

Single Spore ◽

Mycelium Growth ◽

First Report ◽

Tubulin Genes ◽

Agar Plug

In November 2010, four grapevine plants of cv. Crimson from a vineyard located in Sevilla (south Spain) revealed trunk cankers. Several pathogens were isolated, including Cylindrocarpon liriodendri (2), Phaeoacremonium aleophilum (2), Pleurostomophora richardsiae, Neofusicoccum parvum, and Botryosphaeria dothidea (2). Among Botryosphaeriaceae fungi isolated on potato dextrose agar (PDA) were two types that did not fit the above mentioned species. Isolates of type 1 produced an abundant, gray mycelium with a diurnal zonation that gradually became dark olivaceous. Mycelium growth occurred from 5 to 37°C with an optimum at 28°C. Conidia were hyaline, fusiform, aseptate, thin walled, but gradually became obscured and septate with age, and measured (18.4-) 21.4 (-24.3) × (4.2-) 5.5 (-7.2) μm with a length/width (L/W) ratio of 4.0 ± 0.5 (n = 100). Isolates of type 1 were identified as N. mediterraneum (3). Single-spore cultures of type 2 developed a whitish, dense, aerial mycelium and remained white up to 10 days on PDA and darkened to gray thereafter. Mycelium growth occurred from 3 to 37°C with an optimum at 29 to 30°C. Conidia were hyaline, aseptate, thick walled, oblong to cylindrical, sometimes becoming light brown and one or two septate after discharge, and measured (24.6-) 30.2 (-42.8) × (10.9-) 14.3 (-18.6) μm with a L/W ratio of 2.1 ± 0.2 (n =100). Isolates of type 2 were identified as Diplodia corticola (1). Nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region and the -tubulin genes were used to confirm the identifications through BLAST searches in GenBank. Comparison of the sequences of types 1 and 2 showed 99 to 100% homology with N. mediterraneum (HM443604 (4) and GU251836) and D. corticola (AY268421 (1) and EU673117), respectively. Representative sequences of N. mediterraneum (JF949757 and JF949756) and D. corticola (JF949758 and JF949759) were deposited in GenBank. The pathogenicity of one representative isolate of each of N. mediterraneum and D. corticola was confirmed by inoculating 10 detached grapevine canes (averaging 12 mm in diameter and 30 cm long) per isolate. A shallow wound was made with a scalpel on the internodes. A colonized 6-mm agar plug, from the margin of an actively growing colony, was inserted in every wound and sealed with Parafilm. Ten grapevine canes controls received only sterile PDA agar plugs. Canes were maintained at 25°C and 70% humidity. After 5 weeks, all inoculated canes developed cankers and pycnidia around the inoculation site. Vascular necroses that developed on the inoculated canes were an average of 28.6 mm for N. mediterraneum and 27.7 mm for D. corticola. One-way analysis of variance and Tukey's test confirmed significant differences in the extent of vascular necroses. The average necroses length in the inoculated canes was significantly greater (P < 0.05) than the average length of discoloration induced by the simulated inoculation process in the control. Both pathogens were reisolated from all inoculated plants but not from controls. To our knowledge, this is the first report of N. mediterraneum and D. corticola as pathogens on grapevine in Spain. References: (1) A. Alves et al. Mycologia 96:603, 2004. (2) A. Aroca and D. Gramaje et al. Eur. J. Plant. Pathol. 126:165, 2010. (3) P. W. Crous et al. Fungal Planet. No. 19, 2007. (4) F. P. Trouillas et al. Plant. Dis. 94:1267, 2010.

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First Report of Neofusicoccum australe and N. luteum Associated with Canker and Dieback of Quercus robur in Portugal

Plant Disease ◽

10.1094/pdis-10-12-0955-pdn ◽

2013 ◽

Vol 97 (4) ◽

pp. 560-560 ◽

Cited By ~ 11

Author(s):

C. Barradas ◽

A. Correia ◽

A. Alves

Keyword(s):

Quercus Robur ◽

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Deciduous Tree ◽

Pedunculate Oak ◽

Single Spore ◽

Canker Disease ◽

First Report ◽

Wide Range

Quercus robur L., commonly known as “pedunculate oak,” is a long-lived deciduous tree native to most of Europe. It is of great ecological and forestry importance. It is also commonly cultivated as an ornamental tree in parks and gardens. Since 2009 and most likely related to increased drought periods, diseased ornamental trees have been observed in the campus of the University of Aveiro, Portugal. More than 50% of the trees are already damaged by the disease. The symptoms included twig and branch dieback and sunken necrotic bark lesions that could progress to the trunk, resulting in the death of large sections of the tree. Ascomata and conidiomata typical of Botryosphaeriaceae were observed on branches of symptomatic trees. Ascospores were hyaline, aseptate, ovoid to fusoid, and conidia were hyaline, aseptate, smooth, thin-walled, and fusiform with base truncate. Single spore isolates were obtained from samples. In culture, single ascospore isolates produced conidia similar to the ones found on the host. Diseased branch tissues were surface sterilized with 5% NaOCl, plated on potato dextrose agar (PDA), and incubated at 25°C. Fungal isolates recovered produced white aerial mycelium that darkened with age becoming grey to dark grey and conidia that were similar in all aspects to the ones produced by single spore isolates. All isolates produced on PDA a yellow pigment that diffused into the agar and disappeared after 6 to 7 days. Morphological and cultural aspects of the isolates were similar to the species Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips and N. australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (2). Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR fingerprinting divided the isolates into two groups (1). Two isolates (CAA352 and CAA392), one from each group, were selected for further identification by sequencing of the internal transcribed spacer (ITS) region of the rDNA and part of the elongation factor 1-alpha gene (EF1-α) (Accession No. ITS: JX975212 JX975213; EF1-α: JX975210 JX975211). BLAST analysis showed that ITS and EF1-α sequences from group 1 and 2 had 99 to 100% similarity to reference cultures (including ex-type) of N. luteum and N. australe, respectively. To confirm pathogenicity and fulfill Koch's postulates, six 2-year-old seedlings of Q. robur were artificially infected with isolates CAA352 and CAA392 and kept at approximately 20 to 25°C. A shallow wound was done with a scalpel on the basal part of the stem of each seedling, a bark portion was removed aseptically and a PDA disc (0.5 cm) of an actively growing culture was placed on the wound. Control seedlings received sterile PDA discs. The inoculation site was wrapped in Parafilm to prevent desiccation. Within 8 weeks, infected seedlings developed canker lesions associated with vascular necrosis around the inoculation point. A third of the seedlings died and developed abundant pycnidia on the stem. Control seedlings remained symptomless. Both pathogens were successfully reisolated from the infected tissue. N. luteum and N. australe are increasingly reported as causing diseases to a wide range of woody hosts of economic and forestry importance (3). To our knowledge, this is the first report of both species causing dieback and canker disease on Q. robur. References: (1) A. Alves et al. Res. Microbiol. 158:112, 2007. (2) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (3) B. Slippers et al. Fungal Biol. Rev. 21:90, 2007.

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First Report of Neofusicoccum parvum Associated with Grapevine Trunk Diseases in Croatia

Plant Disease ◽

10.1094/pdis-03-13-0283-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1656-1656 ◽

Cited By ~ 6

Author(s):

J. Kaliternam ◽

T. Milicevic ◽

D. Bencic ◽

B. Duralija

Keyword(s):

Aerial Mycelium ◽

Morphological Data ◽

Width Ratio ◽

Vitis Vinifera L ◽

Internal Transcribed Spacer Region ◽

Foeniculum Vulgare ◽

Neofusicoccum Parvum ◽

First Report ◽

Grapevine Trunk Diseases ◽

Trunk Diseases

In September 2010, during survey of diseased grapevines (Vitis vinifera L.) in vineyards at localities Zmajevac (BZ), Orahovica (SO), Cilipi (KC), and Novalja (PN), symptoms characteristic of grapevine trunk diseases (GTD) (3) were observed, showing on cross-sectioned cordons and trunks as brown, wedge-shaped perennial cankers and/or dark streaking of the wood. In Croatia, these symptoms were traditionally associated with Eutypa Tul. & C.Tul. and with fungi from Diaporthaceae (2). From affected grapevines (cvs. Grasevina, Pinot bijeli, Malvazija dubrovacka, and Gegic), samples of symptomatic cordons and trunks were collected (n ≥ 35). To isolate the causal agents from the samples, woodchips of symptomatic tissue, surface-sterilized in 2% sodium hypochlorite for 2 min, were placed on potato dextrose agar amended with streptomycin sulphate (50 μg/ml) and incubated for 7 days at 25°C in darkness. A percentage of samples (72, 15, 27, and 54% from BZ, SO, KC, and PN, respectively) yielded fungal colonies with abundant aerial mycelium, initially white, but turning olivaceous grey after 5 days. From these colonies, monohyphal isolates were obtained and pycnidial formation stimulated by cultivation on 2% water agar with stems of plant species Foeniculum vulgare Mill. at 25°C under diffuse light for 3 weeks. Pycnidia contained conidia that were hyaline, unicellular, ellipsoid with round apices and truncated bases, and thin walled with smooth surface. Dimensions of conidia (n ≥ 50) were (12.8) 15.3 ± 1.4 (17.6) × (5.4) 6.3 ± 0.8 (7.6) μm, with length/width ratio (2.0) 2.5 ± 0.5 (3.2). Based on morphological data, species Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips was suspected (1). For molecular identification, isolates BZ330, SO334, KC342, and PN121 were used for PCR to amplify internal transcribed spacer region and partial translation elongation factor 1-alpha gene, using primers ITS5/ITS4 and EF1-728F/EF1-986R, respectively. Obtained sequences were shown to be identical between the four isolates (GenBank: KF296318, KF296319) and when compared with sequences for reference N. parvum isolate CMW9080 (AY236942, AY236887) they showed >99% homology, confirming the isolates as species N. parvum. Pathogenicity tests were done by inoculation of detached green shoots (GS) and lignified canes (LC) (n = 5) of grapevine cv. Skrlet by either mycelial plugs of the same four isolates, or sterile agar plugs for the controls. Inoculated GS were kept in flasks with sterile water in a glasshouse for 10 days, and LC in humid dark chambers for 30 days, at 25°C. Resulting vascular necrosis measured 62 to 81 mm (GS) and 215 to 246 mm (LC), but was absent on controls. Koch's postulates were satisfied by successful reisolation of N. parvum only from plants inoculated with mycelial plugs. N. parvum has been recognized as a serious grapevine pathogen, causing similar symptoms worldwide (3). To our knowledge, this is the first report of N. parvum associated with GTD in Croatia, and due to its relatively high incidence at surveyed localities, it could present considerable threat, particularly for neighboring vine growing regions. Diplodia seriata De Not., a weak pathogen (3), was also identified from a percentage of samples in this survey. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) J. Kaliterna et al. Arh. Hig. Rada Toksikol. 63:471, 2012. (3) J. R. Urbez-Torres. Phytopathol. Mediterr. 50(Suppl.):S5, 2011.

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First Report of Euphorbia larica Dieback Caused by Fusarium brachygibbosum in Oman

Plant Disease ◽

10.1094/pdis-09-12-0828-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 687-687 ◽

Cited By ~ 9

Author(s):

I. H. Al-Mahmooli ◽

Y. S. Al-Bahri ◽

A. M. Al-Sadi ◽

M. L. Deadman

Keyword(s):

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Common Component ◽

First Report ◽

Ethnobotanical Uses ◽

Artificial Inoculations ◽

Stem Lesions ◽

Desert Flora

Euphorbia larica Boiss. (Arabic = Isbaq) is a dominant and common component of the native desert flora of northern Oman. Traditional ethnobotanical uses have included use of the latex for treating camels with parasites. In February 2011, E. larica plants showing stem lesions up to several cm long and in many cases with stem dieback were collected from Al-Khoudh 50 km west of Muscat. The disease appeared widespread within the location where several dead specimens were also recorded, although the cause was unclear. Sections (5 mm) of five diseased branches taken from different plants and placed on potato dextrose agar (PDA) in all cases yielded Fusarium-like colonies. Colonies recovered were initially white becoming rose to medium red in color with abundant aerial mycelium. Macroconidia were scarce and scattered (mean of 20 spores: 26.83 × 4.73 μm) with three to four septa per spore; microconidia were slightly curved, ovoid, and fusiform (mean of 20 spores: 11.64 × 4.03 μm) with zero to two septa per spore. Spherical chlamydospores (mean of 20 spores: 11.05 μm) were terminal and intercalary, single, and in chains. In vitro characters and spores measurements conformed to previously described features of Fusarium brachygibbosum Padwick (1). Mycelial plugs (5 mm) were taken from 7-day-old cultures of the fungus grown on 2.5% PDA and applied to a small incision (3 mm) on the stems of healthy E. larica grown in situ and protected with wet cotton and Parafilm. The residual agar, mycelium, cotton, and Parafilm were removed after 7 days and symptoms were recorded. Control stems were inoculated using PDA (5 mm) plugs alone and inoculations were repeated twice. Artificial inoculations resulted in dieback of all stems within 11 days and fungal colonies identical to initial isolations were recovered from artificially infected surface-sterilized stem pieces. Identification of F. brachygibbosum was confirmed by comparing sequences generated from the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1 and ITS4 primers) and the intron region of translation elongation factor alpha (EF1-α) (EF-1-986 and EF-728 primers). The ITS and EF1-α sequences were found to share 100% and 99% nucleotide similarity to previously published sequences of the ITS (HQ443206) and EF1-α (JQ429370) regions of F. brachygibbosum in GenBank. The accession number of ITS sequence of one isolate assigned to EMBL-Bank was HF562936. The EF sequence was assigned to EMBL-Bank accession (submission number Hx2000027017; number will be sent later). This pathogen has previously been reported on date palm (2) in Oman but, to our knowledge, this is the first report of this pathogen on E. larica. References: (1) A. M. Al-Sadi et al. Crop Prot. 37:1, 2012. (2) G. W. Padwick. Mycol. Pap. 12:11, 1945.

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First Report of Diaporthe australafricana Associated with Stem Canker on Blueberry in Chile

Plant Disease ◽

10.1094/pdis-12-11-1025-pdn ◽

2012 ◽

Vol 96 (5) ◽

pp. 768-768 ◽

Cited By ~ 3

Author(s):

B. A. Latorre ◽

K. Elfar ◽

J. G. Espinoza ◽

R. Torres ◽

G. A. Díaz

Keyword(s):

Vitis Vinifera ◽

Vascular Tissue ◽

Its Region ◽

Pathogenic Fungi ◽

Stem Canker ◽

Vaccinium Corymbosum ◽

Academic Press ◽

First Report ◽

Potted Plants ◽

Blastn Analysis

Stem cankers of blueberry (Vaccinium corymbosum L.) have been observed on as much as 15% of the plants in plantations in central and southern Chile since 2006. Symptoms consisted of apical necrosis of the shoots and brown-to-reddish necrotic lesions on the stems. Internally, a brown-to-reddish discoloration of the vascular tissue can be observed. Twenty, single-plant samples were collected in 12 blueberry plantings (approximately 33°27′ to 40°53′S). Isolations from the margins of the necrotic lesions on the stems were made by plating small pieces (5 mm) on potato dextrose agar acidified with 0.5 μl/ml of 92% lactic acid (APDA). The plates were incubated at 20°C for 5 to 7 days, and hyphal tips of white colonies with septate and hyaline mycelium were transferred to APDA. Colonies were then transferred to autoclaved Pinus radiata needles on 2% water agar and incubated for 20 days at 20°C. Twelve isolates producing black pycnidia and alpha conidia were tentatively identified as a Phomopsis sp. (teleomoph Diaporthe Nitschke). Other fungi, including Botryosphaeriaceae spp. and Pestalotiopsis spp., were also isolated. Alpha conidia were smooth, unicellular, hyaline, fusoid, biguttulate, and 6.4 to 7.9 × 2.3 to 3.3 μm (n = 20). Beta conidia were not observed. The internal transcribed spacer (ITS) region of the rDNA was amplified using primers ITS1 and ITS2 (4) and sequenced. BLASTn analysis of the 473-bp fragment (GenBank Accession No. JQ045712) showed 100% identity to Diaporthe australafricana Crous & J.M. van Niekerk from Vitis vinifera (3). The pathogenicity of D. australafricana was studied on blueberry cv. O'Neal using detached stems (n = 4) in the laboratory, on 2-year-old potted plants (n = 4) in a greenhouse, and on attached stems of mature plants (n = 4) established in the ground. Inoculations were done by placing mycelial plugs taken from 7-day-old APDA cultures in a 7-mm long incision made on the stems. Inoculations with sterile mycelium plugs served as negative controls. Inoculation sites were wrapped with Parafilm to avoid rapid dehydration. Dark brown, necrotic lesions on the internal tissues were obtained on all inoculated stems 15 days after inoculation. Mean lesion lengths were 18.0 ± 7.4 mm on detached stems, 7.8 ± 6.9 mm on stems of 2-year-old plants, and 7.3 ± 2.5 mm on mature plants in the field. No symptoms developed on control stems. Reisolations were successful in 100% of the inoculated stems and D. australafricana was confirmed by the presence of pycnidia and alpha conidia. To our knowledge, this is the first report of D. australafricana causing stem canker in V. corymbosum. Previously, this pathogen has been reported to be affecting Vitis vinifera in Australia and South Africa (3). These results do not exclude that other plant-pathogenic fungi may be involved in this syndrome (1,2). References: (1) J. G. Espinoza et al. Plant Dis 92:1407, 2008. (2) J. G. Espinoza et al. Plant Dis. 93:1187, 2009. (3) J. M. van Niekerk et al. Australas. Plant Pathol. 34:27, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, NY, 1990.

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First Report of Lasiodiplodia theobromae Associated with Decline of Grapevine Rootstock Mother Plants in Spain

Plant Disease ◽

10.1094/pdis-92-5-0832b ◽

2008 ◽

Vol 92 (5) ◽

pp. 832-832 ◽

Cited By ~ 13

Author(s):

A. Aroca ◽

R. Raposo ◽

D. Gramaje ◽

J. Armengol ◽

S. Martos ◽

...

Keyword(s):

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Pathogenic Fungi ◽

Vascular Lesions ◽

Malt Extract ◽

Pathogenicity Test ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Mother Plants

A field of Richter 110 rootstock mother plants in Valencia Province (eastern Spain) was surveyed during November 2006 to study the mycoflora of declining plants. Two canes with stunted leaves were collected from a plant with a reduced number of shoots. No cankers or vascular lesions were observed in the collected canes. Six wood chips (1 to 2 mm thick) were taken from one basal fragment (3 to 4 cm long) of each cane, surface sterilized in 70% ethanol for 1 min, and plated on malt extract agar supplemented with 0.5 g L–1 of streptomycin sulfate. Petri dishes were incubated for 7 days at 25°C. A fungus was consistently isolated from all samples that showed the following characteristics: colonies grown on potato dextrose agar (PDA) at 25°C developed a white, aerial mycelium that turned gray after 4 to 6 days and produced pycnidia after 1 month on sterile grapevine slivers of twigs placed on the PDA surface; conidia from culture were ellipsoidal, thick walled, initially hyaline, nonseptate, and measuring 20 to 25 (22.5) × 12 to 14 (13) μm; aged conidia were brown, 1-septate with longitudinal striations in the wall; and pseudoparaphyses variable in form and length were interspersed within the fertile tissue. The fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the above characteristics (2). Identity was confirmed by analysis of the nucleotide sequences of the internal transcribed spacer (ITS) region from the rRNA repeat and part of the translation elongation factor 1-alpha (EF1-α) and the β-tubulin (B-tub) genes, as done elsewhere (1,3). BLAST searches at GenBank showed a high identity with reference sequences (ITS: 100%, EF1-α: 97%; B-tub: 99%). Representative sequences of the studied DNA regions were deposited at GenBank (Accession Nos.: ITS: EU254718; EF1-α: EU254719; and B-tub: EU254720). A pathogenicity test was conducted on 1-year-old grapevine plants cv. Macabeo grafted onto Richter 110 rootstocks maintained in a greenhouse. A superficial wound was made on the bark of 10 plants with a sterilized scalpel, ≈10 cm above the graft union. A mycelial plug obtained from the margin of an actively growing fungal colony (isolate JL664) was placed in the wound and the wound was wrapped with Parafilm. Ten additional control plants were inoculated with sterile PDA plugs. All control plants grew normally, and the inoculation wound healed 3 months after inoculation. Plants inoculated with L. theobromae showed no foliar symptoms in the same period, but developed cankers variable in size surrounding the inoculation sites. Vascular necroses measuring 8.4 ± 1.5 cm (mean ± standard error) developed in the inoculated plants that were significantly longer than the controls (0.3 ± 0.2 cm). The pathogen was reisolated from all inoculated plants and no fungus was reisolated from the controls. These results confirmed the pathogenicity of L. theobromae to grapevine and points to a possible involvement of L. theobromae in the aetiology of grapevine decline as previously reported (3,4). To our knowledge, this is the first report of L. theobromae isolated from grapevine in Spain. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) E. Punithalingam. No. 519 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1976. (3) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006. (4) J. M. van Niekerk et al. Phytopathol. Mediterr. 45(suppl.):S43, 2006.

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Identification of Neofusicoccum parvum causing canker and twig blight on Ficus carica in Italy

Phytopathologia Mediterranea ◽

10.36253/phyto-10798 ◽

2020 ◽

Vol 59 (1) ◽

pp. 213-218

Author(s):

Dalia AIELLO ◽

Giorgio GUSELLA ◽

Alberto FIORENZA ◽

Vladimiro GUARNACCIA ◽

Giancarlo POLIZZI

Keyword(s):

Sequence Data ◽

Elongation Factor ◽

Its Region ◽

Ficus Carica ◽

Translation Elongation ◽

Neofusicoccum Parvum ◽

Elongation Factor 1 Alpha ◽

Twig Blight ◽

Pathogenicity Tests ◽

Fungal Colony

During June 2018, several symptomatic fig (Ficus carica) cuttings, showing twig blight, subcortical discolouration and apical dieback were collected from a nursery in Catania province, Sicily (Italy). Isolations from diseased tissue consistently showed the presence of the same fungal colony. Morphology of the fungal isolates together with sequence data of the nuclear rDNA internal transcriber spacer (ITS) region, translation elongation factor 1-alpha (tef1) gene and partial beta-tubulin (tub2) gene of representatives isolates revealed the presence of the fungus Neofusicoccum parvum. Pathogenicity tests were conducted by inoculating fig cuttings with mycelial plugs. After 10 days, the inoculated plants developed cankers similar to those observed in the greenhouse and after 26 days all inoculated plants were dead. To the best of our knowledge, this is the first report worldwide of N. parvum causing disease on this host.

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Identification and pathogenicity of Diplodia, Neofusicoccum, Cadophora and Diaporthe species associated with cordon dieback in kiwifruit cv. Hayward in central Chile

Plant Disease ◽

10.1094/pdis-05-20-0988-re ◽

2020 ◽

Author(s):

Gonzalo A Díaz ◽

Juan Pablo Zoffoli ◽

Enrique Ferrada ◽

Mauricio A. Lolas

Keyword(s):

Elongation Factor ◽

Its Region ◽

Fruit Trees ◽

Economic Losses ◽

Translation Elongation ◽

Diplodia Seriata ◽

Neofusicoccum Parvum ◽

Central Chile ◽

Field Surveys ◽

Dieback Disease

Dieback symptoms associated with fungal trunk pathogens cause significant economic losses to kiwifruit and other woody fruit trees worldwide. This study represents the first attempt to identify and characterize the fungal trunk pathogens associated with cordon dieback disease of kiwifruit in central Chile. Field surveys were conducted throughout the main kiwifruit-growing regions in central Chile to determine the incidence and to characterize the fungal trunk pathogens associated with cordon dieback of kiwifruit cv. Hayward through morphological, molecular and pathogenicity studies. A total of 250 cordon samples were collected, and the isolations were performed on acidified potato dextrose agar (2%, APDA) plus antibiotics and Igepal. The incidence of kiwifruit cordon dieback ranged between 5 and 85% in all surveyed areas in central Chile. A total of 246 isolates were isolated and identified using culture and morphological features as belonging to three fungal taxa. Diaporthaceae spp. (Diaporthe ambigua and D. australafricana; n=133 isolates), Botryosphaeriaceae spp. (Diplodia seriata and Neofusicoccum parvum; n=89 isolates) and Ploettnerulaceae spp. (Cadophora luteo-olivacea and C. malorum; n=24 isolates) were identified using phylogenetics studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the β-tubulin gene (tub2) and part of the translation elongation factor 1-α gene (tef1-α). Isolates of N. parvum and Di. seriata were the most virulent, causing internal brown lesion and dieback symptoms in attached green shoots, attached lignified canes and young inoculated kiwifruits. This report is the first to describe Di. seriata and C. luteo-olivacea associated with kiwifruit cordon dieback in Chile and presents the first description of N. parvum causing kiwifruit dieback worldwide.

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Valdensinia heterodoxa Peyronel as a New Pathogen of Blueberry in Poland

Plant Disease ◽

10.1094/pdis-06-13-0644-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 688-688

Author(s):

R. Dzięcioł ◽

E. Mirzwa-Mróz ◽

E. Zielińska ◽

M. Wińska-Krysiak ◽

W. Wakuliński

Keyword(s):

Soil Surface ◽

Its Region ◽

Vaccinium Corymbosum ◽

Leaf Blight ◽

Single Spore ◽

Initial Symptoms ◽

Sterile Needle ◽

Young Leaves ◽

Necrotic Spots ◽

Thermal Cycler

Valdensia leaf blight on blueberry in Poland was reported in one commercial nursery plantation near Prażmów, Mazovia voivodship, where heavy defoliation was observed on cv. Bluecrop, grown in nursery pots, in August 2011. Older fruiting bushes were only slightly affected by the disease. Initial symptoms of the disease were small, oval to circular zonated necrosis surrounded with dark brown borders that enlarged on the leaves throughout the canopy. Multicellular, hyaline or light brown, star-shaped conidiospores were observed on the necrotic areas. The mean length of 50 conidiospores from the end of head to the end of arm apex was 307 to 348 μm (4). Eight single-spore isolates of the fungus were obtained. Single conidiospores were picked up from necrotic spots on leaves and transferred with sterile needle on potato dextrose agar (PDA) and incubated at 20°C under ambient light. After 10 days of incubation, total DNA was extracted. Amplification of the internal transcribed spacer (ITS) region of rDNA was done using primers ITS1F and ITS4A (1). PCRs were carried out as follows: initial denaturation at 94°C for 2 min, denaturation at 94°C for 1 min, annealing at 57°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min for 28 cycles (Applied Biosystems Veriti 96 Wel Thermal Cycler). Amplicons, which were approximately 520 bp, were sequenced and nucleotide sequences were analyzed by Clustal W2EBI. The sequences of all eight isolates showed 100% similarity to each other and were compared with sequences stored in GenBank using BLAST. Sequences were 525 bp long and showed 100% homology to Valdensinia heterodoxa Peyronel, Sclerotiniaceae (anamorph: Valdensia heterodoxa Peyronel) from Japan and Norway (Accession Nos. AB663682 and Z81447, respectively) (3). The sequence from one isolate was submitted to GenBank (Accession No. KF212190). To fulfill Koch's postulates, each of the eight isolates was used to inoculate 20 healthy young leaves of Vaccinium corymbosum L. cv. Bluecrop and bilberry (V. myrtillus L.) (10 leaves per plant). Mycelial plugs 5 mm in diameter were taken from PDA cultures, approximately 20 days old, and used as inoculum and placed in the center of each leaf and moistened with sterile distilled water. Mycelium-free plugs were used as control. Inoculated leaves were placed in plastic box and incubated at 20°C in laboratory for 5 days, at which time small necrotic lesions consistent with initial symptoms of the disease were observed. Isolates obtained from these symptoms were morphologically identical to those used for inoculation. Control leaves did not show any disease symptoms. Valdensia leaf blight occurrence may be attributed to rainy July and August 2011 and long presence of water on soil surface. In Poland, Valdensinia heterodoxa causes heavy defoliation of Vaccinium myrtillus in pine stands and is a common pathogen of some herbaceous plants (2). To our knowledge, this is the first report of Valdensia leaf blight on highbush blueberry in Poland. References: (1) I. Larena et al. 75:187, 1999. (2) W. Mułenko and S. Woodward. Mycologist 10:69, 1996. (3) S. Nekoduka et al. J. Gen. Plant Pathol. 78:151, 2012. (4) S. Zhao and S. F. Shamoun. Mycology 1:113, 2010.

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