David Gramaje

Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain)

Severe decline of almond trees has recently been observed in several orchards on the island of Mallorca (Balearic Islands, western Mediterranean Sea). However, the identity of the causal agents has not yet been investigated. Between August 2008 and June 2010, wood samples from branches of almond trees showing internal necroses and brown to black vascular streaking were collected in the Llevant region on the island of Mallorca. Several fungal species were subsequently isolated from the margin between healthy and symptomatic tissue. Five species of Botryosphaeriaceae (namely Botryosphaeria dothidea, Diplodia olivarum, D. seriata, Neofusicoccum australe and N. parvum), Eutypa lata, Phaeoacremonium iranianum and Phomopsis amygdali were identified based on morphology, culture characteristics and DNA sequence comparisons. Neofusicoccum parvum was the dominant species, followed by E. lata, D. olivarum and N. australe. First reports from almond include D. olivarum and Pm. iranianum. Two species are newly described, namely Collophora hispanica sp. nov. and Phaeoacremonium amygdalinum sp. nov.

Evaluation of the grapevine nursery propagation process as a source of Phaeoacremonium spp. and Phaeomoniella chlamydospora and occurrence of trunk disease pathogens in rootstock mother vines in Spain

Five commercial nurseries were sampled in 2007 to evaluate the grapevine nursery propagation process as a source of Petri disease pathogens (Phaeoacremonium spp. and Phaeomoniella chlamydospora). Samples were taken at four stages of the propagation process: pre-grafting hydration tanks, scissors used for cutting buds, grafting machines and peat used to promote root development. All samples were analysed using two different techniques: nested PCR using specific primers for Phaeoacremonium spp. (Pm1/Pm2) and Pa. chlamydospora (Pch1/Pch2); and fungal isolation by culturing on semi-selective medium. Either Phaeoacremonium spp. or Pa. chlamydospora were detected at any of these stages, and more importantly they were viable since they were detected by isolating on culturing medium. Additionally, the importance of grapevine rootstock mother fields as sources of inoculum in the nurseries was studied. Fourteen grapevine rootstock mother fields were surveyed in 2006 and 2007 for the occurrence of fungal trunk pathogens. A total of 16.4% and 30% of the plants sampled in 2006 and 2007, respectively were infected. Petri disease pathogens (Pa. chlamydospora, Phaeoacremonium aleophilum, Pm. parasiticum) and several Botryosphaeriaceae species (Neofusicoccum parvum, Botryosphaeria dothidea, Lasiodiplodia theobromae, N. australe, N. mediterraneum and N. vitifusiforme) and Phomopsis viticola were isolated. This is the first time N. mediterraneum has been isolated from grapevines and the first report of N. australe, N. mediterraneum and N. vitifusiforme in Spain. This work shows that grapevine rootstock mother plants and the propagation process of grapevine plants should be considered as important sources of inoculum for fungal trunk pathogens, and especially of Petri disease pathogens.

Phaeoacremonium: from esca disease to phaeohyphomycosis.

Phaeoacremonium spp. are commonly isolated from stems and branches of diseased woody hosts, and humans with phaeohyphomycosis. The genus Phaeoacremonium (Togniniaceae, Togniniales) has recently been monographed, and presently contains 46 species, while its sexual morph, Togninia, contains 26 epithets, of which 13 are insufficiently known. In this review we summarise information pertaining to the global distribution, pathology, ecology, and detection of these species, and present a case for retaining the genus Phaeoacremonium over that of Togninia. Furthermore, to obtain a single nomenclature, the following new combinations are also proposed: Phaeoacremonium africanum, P. aquaticum, P. fraxinopennsylvanicum, P. griseo-olivaceum, P. inconspicuum, P. leptorrhynchum, P. minimum, and P. vibratile.

Evaluation of Vineyard Weeds as Potential Hosts of Black-Foot and Petri Disease Pathogens

Weeds were sampled in grapevine rootstock mother fields, open-root field nurseries, and commercial vineyards of Albacete, Alicante, Castellón, Murcia, and Valencia provinces in Spain between June 2009 and June 2010 and evaluated as potential hosts of black-foot and Petri disease pathogens. Isolations were conducted in the root system and internal xylem tissues for black-foot and Petri disease pathogens, respectively. Cylindrocarpon macrodidymum was successfully isolated from the roots of 15 of 19 weed families evaluated and 26 of 52 weed species. Regarding Petri disease pathogens, one isolate of Phaeomoniella chlamydospora was obtained from Convolvulus arvensis, and three isolates of Cadophora luteo-olivacea were obtained from Bidens subalternans, Plantago coronopus, and Sonchus oleraceus. Pathogenicity tests showed that Cylindrocarpon macrodidymum isolates obtained from weeds were able to induce typical black-foot disease symptoms. When inoculated in grapevines, isolates of Cadophora luteo-olivacea and Phaeomoniella chlamydospora were also shown to be pathogenic on grapevine cuttings. Our ability to recover grapevine pathogens from vineyard weeds and to demonstrate pathogenicity of recovered strains on grape suggests that these weeds may serve as a source of inoculum for infection of grapevine.

Field Evaluation of Grapevine Rootstocks Inoculated with Fungi Associated with Petri Disease and Esca

One-year-old grapevine rootstock cuttings of 41B Millardet Grasset, 140 Ruggeri, 161-49 Couderc, 1103 Paulsen, and 110 Richter were inoculated with pathogens associated with Petri disease and esca of grapevine to determine the effects of fungal infection on percentage of cuttings emerging from dormancy, shoot weight, and disease severity. The cuttings were vacuum-inoculated with spore suspensions of either Cadophora luteo-olivacea, five species of Phaeoacremonium, or Phaeomoniella chlamydospora and planted in two field sites in March 2008. Most of the fungal pathogens caused a significant reduction in the percentage of cuttings emerging from dormancy and shoot weight and a significant increase in disease severity in all grapevine rootstocks except 161-49 Couderc. Rootstocks 110 Richter and 140 Ruggeri were greatly affected by fungi associated with Petri disease and esca. In general, Pa. chlamydospora and Pm. parasiticum caused the greatest reduction in percentage of cuttings emerging from dormancy and shoot weight and the highest increase in disease severity. Regression analyses showed a significant correlation between percentage of cuttings emerging from dormancy and disease severity and between shoot weight and disease severity in almost all rootstocks inoculated with Pa. chlamydospora.

Detection of black-foot and Petri disease pathogens in soils of grapevine nurseries and vineyards using bait plants

Background and aimsLittle information is currently available regarding the number of species of black-foot and Petri disease pathogens present in soil and their capacity to infect grapevine roots and reach the xylem vessels.MethodsSeedlings of grapevine rootstock 41-B, and cvs. Bobal and Palomino were planted both in pots containing soil samples collected from commercial vineyards and in nursery fields. Roots and xylem vessels were later analyzed for fungal isolation.ResultsBlack-foot pathogens: Ilyonectria alcacerensis, I. macrodidyma, I. novozelandica and I. torresensis were frequently isolated from roots of seedlings grown in all soils evaluated, whereas Petri disease pathogens: Cadophora luteo-olivacea, Phaeoacremonium aleophilum, Pm. parasiticum and Phaeomoniella chlamydospora were only isolated from xylem vessels of seedlings grown in nursery soils, with a low incidence. Ilyonectria alcacerensis, I. novozelandica and I. torresensis were isolated for the first time from grapevines in Spain, and Pm. parasiticum and Ca. luteo-olivacea were detected for the first time in nursery soils.ConclusionsOur results confirm nursery and vineyard soils as an important inoculum source for black-foot pathogens and demonstrate the presence of several Petri disease pathogens in nursery soils.

Multilocus ISSR Markers Reveal Two Major Genetic Groups in Spanish and South African Populations of the Grapevine Fungal Pathogen Cadophora luteo-olivacea

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions.

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

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.

First Report of Phaeoacremonium krajdenii Causing Petri Disease of Grapevine in Spain

In September 2009, symptoms of grapevine (Vitis vinifera L.) decline were observed on 3-year-old grapevines in a vineyard in Roquetas de Mar (Almeria Province, southern Spain). Affected vines were weak with reduced foliage and chlorotic leaves. Black spots and dark streaking of the xylem vessels could be seen in cross- or longitudinal sections of the rootstock trunk. Symptomatic plants were collected and sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Dishes were incubated at 25 to 26°C in the dark for 14 to 21 days, and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (2). Colonies were grayish brown on PDA and dark brown on MEA. Conidiophores were short and unbranched and 11.5 to 46 (25.5) μm long. Phialides were often polyphialidic. Conidia were hyaline, oblong-ellipsoidal or allantoid, 2.5 to 5 (4.2) μm long, and 1 to 1.7 (1.2) μm wide. On the basis of these characters, the isolates were identified as Phaeoacremonium krajdenii L. Mostert, Summerb. & Crous (1,2). DNA sequencing of a fragment of the beta-tubulin gene of the isolate (Pkr-1) using primers T1 and Bt2b (GenBank Accession No. HM637892) matched P. krajdenii GenBank Accession No. AY579330. Pathogenicity tests were conducted using isolate Pkr-1. Ten 1-year-old callused and rooted cuttings of 110 R rootstock grown in pots with sterile peat were wounded at the uppermost internode with an 8-mm cork borer. A 5-mm mycelium PDA plug from a 2-week-old culture was placed in the wound before being wrapped with Parafilm. Ten control plants were inoculated with 5-mm noncolonized PDA plugs. Plants were maintained in a greenhouse at 25 to 30°C. Within 3 months, shoots on all Phaeoacremonium-inoculated cuttings had weak growth with small leaves and short internodes and there were black streaks in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.5 ± 1.2 cm long, significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was reisolated from discolored tissue of all inoculated cuttings, completing Kochs postulates. P. krajdenii has a worldwide distribution, although these reports are from human infections (1). P. krajdenii was first reported as a pathogen of grapevines in South Africa (1). To our knowledge, this is the first report of P. krajdenii causing young grapevine decline in Spain or any country in Europe. References: (1) L. Mostert et al. J. Clin. Microbiol. 43:1752, 2005. (2) L. Mostert et al. Stud. Mycol. 54:1, 2006.

New Phaeoacremonium species isolated from sandalwood trees in Western Australia.

Thirty-eight Phaeoacremonium isolates collected from pruning wounds of tropical sandalwood in Western Australia were studied with morphological and cultural characteristics as well as phylogenetic analyses of combined DNA sequences of the actin and β-tubulin genes. Three known Phaeoacremonium species were found, namely P. alvesii, P. parasiticum, and P. venezuelense. Phaeoacremonium venezuelense represents a new record for Australia. Two new species are described: P. luteum sp. nov. can be identified by the ability to produce yellow pigment on MEA, PDA, and OA, the predominance of subcylindrical to subulate type II phialides, and the mycelium showing prominent exudate droplets observed as warts; and P. santali sp. nov. which can be separated from other species producing pink colonies on MEA by the predominance of type I and II phialides, the distinct brownish olive colonies in OA, and slow growth.