Neofusicoccum Mediterraneum Research Articles

Identification, pathogenicity and distribution of the causal agents of dieback in avocado orchards in Spain

An increased incidence of dieback from branches in several avocado orchards in southern Spain was observed in 2014. Surveys were conducted from May to October 2014, sampling the affected branches to isolate the causal agents. A total of 68 fungal isolates, recovered from ten avocado orchards, were identified, by morphological characterisation and DNA sequencing, as belonging to the genera: Neofusicoccum parvum (50%), Colletotrichum gloeosporioides (17.6%), Neofusicoccum luteum (16.2%), Neofusicoccum australe (13.2%), Neofusicoccum mediterraneum (1.5%) and Lasiodiplodia theobromae (1.5%). A decreasing level of virulence in artificial inoculations on avocado plants was observed in N. parvum, N. luteum, N. mediterraneum, N. australe, C. gloeosporioides and L. theobromae, there were significant differences among N. parvum and the rest of species of this genus, and significant differences were only observed between N. luteum and C. gloeosporioides. The geographical distribution of N. parvum and N. Luteum covers different areas, while C. gloeosporioides and N. australe are located only in the areas around Benamocarra and Vélez-Málaga (southern Spain), while N. mediterraneum and L. theobromae appear only occasionally. This is the first study of avocado branch cankers in Spain which identifies the causal agents and establishes their pathogenicity groups, with N. parvum as the most important causal agent of avocado dieback in this area.

Interaction Between Diaporthe rhusicola and Neofusicoccum mediterraneum Causing Branch Dieback and Fruit Blight of English Walnut in California, and the Effect of Pruning Wounds on the Infection.

Botryosphaeriaceae and Diaporthaceae species are the causal agents of branch dieback of English walnut in California. In this study, the effects of the interaction between Neofusicoccum mediterraneum and Diaporthe rhusicola were evaluated in vitro by using mycelial plugs or spore suspensions and in vivo by inoculating shoots and epicarps (hulls) of walnut. Single inoculations of each species and different coinfection treatments were performed under laboratory or field conditions. The influence of shoot age and susceptibility of bark or pith tissues to N. mediterraneum and D. rhusicola infection after pruning was also evaluated. In in vitro experiments, spore germination of D. rhusicola was significantly (P < 0.0001) reduced in the presence of N. mediterraneum spores. When D. rhusicola was inoculated at 4 days before N. mediterraneum, a delay in lesion development in shoots and hulls was observed compared with the other two interaction treatments. One- to 2-year-old shoots were more susceptible to infection and colonization by N. mediterraneum than 3- to 4-year-old shoots. In young shoots, inoculation in the pith tissue resulted in longer lesions than those observed on shoots inoculated in the bark. No significant differences were observed between the development of internal or external necrosis and the age of the shoots, or the susceptibility of bark and pith to D. rhusicola infection. This information is essential to better understanding the complex situation of this walnut disease toward developing control management strategies.

Occurrence of grapevine trunk diseases affecting the native cultivar Pedro Ximénez in southern Spain

‘Pedro Ximenez’ is a native grapevine cultivar from Cordoba province (Andalusia region, southern Spain), which has been seriously affected by fungal trunk pathogens causing general decline along the last decade. However, the occurrence of grapevine trunk diseases (GTDs) in Andalusia region has not been studied yet. Therefore, the main goal of this study was to evaluate the occurrence of GTDs affecting ‘Pedro Ximenez’, and to characterize the causal agents by morphological and molecular methods. Between 2016 and 2017, 25 commercial vineyards of cv. Pedro Ximenez showing decline symptoms were surveyed. Fungal isolations were done and 23 representative isolates of the fungal trunk pathogens associated with the different GTDs (black-foot and Petri disease, Botryosphaeria and eutypa dieback, and esca) were selected according to colony colour and mycelial growth development. Colony colour, mycelial growth, and conidial characteristics were defined. The effect of temperature on mycelial growth was evaluated. Phylogenetic analyses of ribosomal genes (ITS) and functional protein regions (ACT, HIS, TEF, TUB) were preformed to confirm their identification. The especies Cadophora luteo-olivacea, Cryptovalsa ampelina, Dactylonectria alcacerensis, Da. novozelandica, Diplodia corticola, D. mutila, D. seriata, Eutypa lata, Fomitiporia mediterranea, Ilyonectria liriodendri, Neofusicoccum mediterraneum, N. parvum, Phaeoacremonium iranianum, Pm. minimum and Phaeomoniella chlamydospora were identified. Despite grapevine fungal trunk pathogens being well known in the main grape growing areas worldwide, this present study is relevant because it has been focused in the native cultivar Pedro Ximenez, improving our knowledge about aetiology of GTDs in southern Spain towards make decisions about management strategies preventing infections.

Effects of Wound Size, Amount of Sap, and Number of Blighted Nuts on Infection of Pistachio Organs by Neofusicoccum mediterraneum

Laboratory and field studies were conducted to determine the effects of wounding of nut exocarp, susceptibility period after wounding, and sap nut on infection of pistachio nut by Neofusicoccum mediterraneum, the main causal agent of panicle and shoot blight of pistachio. Under controlled conditions and in the field, detached nuts were inoculated with a conidial suspension 30 min before or after wounding. In addition, a 30-µl drop of pistachio sap was placed on the surface of noninjured nuts 30 min before or after they were wounded and then inoculated. Wounding increased the disease severity under both controlled and field conditions. The addition of sap increased the susceptibility of nuts under controlled conditions but not in the field, possibly due to dried sap blocking the pathogen infection. When nuts of Kerman, Kalehghouchi, and Golden Hills pistachio were wounded and inoculated at different time periods after wounding; the nuts of the three cultivars were highly susceptible to pathogen infection during at least the first 24 h after wounding. Under field conditions, there was not a clear effect of increasing the number of inoculated nuts per panicle or the inoculation position (basal or apical) in killing (blight) of the panicle. Conversely, inoculations conducted with mycelial plugs resulted in higher disease, increased the proportion of dead panicles, and resulted in faster symptom expression than inoculations conducted with a conidial suspension. To determine the temporal infection pattern, leaves and panicles were regularly collected from different orchards from 2004 to 2007 and the pathogen was isolated on medium. Important differences in latent infection were detected between years and orchards, with nut and rachis being, in general, the tissues most susceptible to infection. Results of this study help in better understanding the dynamic of infection and colonization of pistachio by N. mediterraneum.

Identification of Fungal Species Associated with Branch Dieback of Olive and Resistance of Table Cultivars to Neofusicoccum mediterraneum and Botryosphaeria dothidea.

Over two consecutive seasons, 16 olive orchards with trees exhibiting dieback symptoms on branches were surveyed in southern Spain. The six dominant fungal species recovered were characterized by means of phenotypic observations, DNA analysis (by sequencing of the internal transcribed spacer, β-tubulin, and large subunit nuclear ribosomal DNA regions), and pathogenicity tests. Additionally, three isolates collected from Tunisian olive trees showing similar dieback symptoms, one isolate of Colletotrichum godetiae, and a reference isolates of Neofusicoccum mediterraneum were included. The resistance of the 11 most important table cultivars to N. mediterraneum and Botryosphaeria dothidea, the causal agent of "escudete" (small shield) of fruit, was studied by the inoculation of branches and immature fruit, respectively. The species Cytospora pruinosa, N. mediterraneum, Nothophoma quercina, Comoclathris incompta, and Diaporthe sp. were identified. Only N. mediterraneum and C. incompta were able to induce the typical dieback symptoms and cankers that affected the development of the plants. The species N. mediterraneum was the most virulent among the evaluated species, although differences in virulence among its isolates were observed. The remaining fungal species were weakly pathogenic to nonpathogenic on plants. According to resistance tests, 'Gordal Sevillana' and 'Manzanilla Cacereña' were the most susceptible to branch dieback caused by N. mediterraneum. Furthermore, the fruit of 'Aloreña de Atarfe' and 'Manzanilla de Sevilla' were the most susceptible to B. dothidea. Knowledge of the etiology and cultivar resistance of these diseases will help to establish better control measures.

Characterization of Botryosphaeriaceae species associated with diseased loquat (Eriobotrya japonica) in Spain

Loquat (Eriobotrya japonica) is an important subtropical fruit crop in Spain and other Mediterranean countries. In recent years, characteristic symptoms of branch canker and dieback have been observed in the main cultivated areas of loquat in Spain. The goal of this study was to identify and characterize the species of Botryosphaeriaceae associated with these symptoms. For this, 36 affected orchards were surveyed between 2010 and 2011 in six provinces of southeastern Spain. Eighty‐four isolates belonging to the family Botryosphaeriaceae were recovered from samples with symptoms. These isolates were characterized by means of phenotypical studies, DNA sequence analyses of the internal transcribed spacer (ITS) and part of the translation elongation factor 1‐α regions, and pathogenicity tests. Ten fungal species were identified: Diplodia malorum, Diplodia olivarum, Diplodia seriata, Diplodia pseudoseriata/Diplodia alatafructa, Diplodia sp., Dothiorella sarmentorum, Neofusicoccum mediterraneum, N. parvum, Spencermartinsia plurivora and S. viticola. In addition, Diplodia eriobotryicola and Dothiorella eriobotryae are newly described. The most frequent species isolated from cankers was D. seriata and, as far as is known, this is the first report of D. malorum, and species belonging to the complex D. pseudoseriata/D. alatafructa, in Spain. All species were pathogenic to 1‐year‐old loquat plants cv. Algerie, with Diplodia sp. and S. viticola as the most virulent.

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.

Phylogeny, Morphology, Distribution, and Pathogenicity of Botryosphaeriaceae and Diaporthaceae from English Walnut in California.

Species of family Botryosphaeriaceae and genus Diaporthe (anamorph: genus Phomopsis, family Diaporthaceae) were reported and caused diseases on various fruit and nut trees in California. In the last several years, diseases on English walnut (Juglans regia) caused by species of Botryosphaeriaceae and Diaporthe were observed frequently in California. Disease symptoms include stem canker; shoot canker and blight; twig, leaf, and fruit blight; and necrotic leaf lesions. Isolates of the pathogen were collected from English walnut in 13 counties in California. The aims of this study were to identify these isolates and to test their pathogenicity to English walnut cultivars. In total, 159 California isolates were identified based on comparisons of DNA sequence data of the internal transcribed spacer, translation elongation factor 1-α, and β-tubulin gene regions, and combined with the morphological features of the cultures and conidia. Research results revealed that isolates represent 10 species of Botryosphaeriaceae and two species of Diaporthe. These species include Botryosphaeria dothidea, Diplodia mutila, D. seriata, Dothiorella iberica, Lasiodiplodia citricola, Neofusicoccum mediterraneum, N. nonquaesitum, N. parvum, N. vitifusiforme, Neoscytalidium dimidiatum, Diaporthe neotheicola, and D. rhusicola. Pathogenicity on three English walnut cultivars ('Chandler', 'Tulare', and 'Vina') using a mycelium plug inoculation method revealed that all these species are pathogenic to all the tested cultivars, with L. citricola and N. parvum being the most pathogenic species, followed by N. mediterraneum, N. dimidiatum, and B. dothidea. Chandler was more tolerant to infection than Tulare and Vina. Results in this study determined that multiple numbers of the Botryosphaeriaceae fungi and two Diaporthe spp. cause cankers and blights of English walnut and vary in their virulence from highly to slightly virulent, respectively.

Botryosphaeriales fungi produce extracellular enzymes with biotechnological potential.

Phytopathogenic fungi are known for producing an arsenal of extracellular enzymes whose involvement in the infection mechanism has been suggested. However, these enzymes are largely unknown and their biotechnological potential also remains poorly understood. In this study, the production and thermostability of extracellular enzymes produced by phytopathogenic Botryosphaeriaceae was investigated. Hydrolytic and oxidative activities were detected and quantified at different temperatures. Most strains (70%; 37/53) were able to produce simultaneously cellulases, laccases, xylanases, pectinases, pectin lyases, amylases, lipases, and proteases. Surprisingly for mesophilic filamentous fungi, several enzymes proved to be thermostable: cellulases from Neofusicoccum mediterraneum CAA 001 and from Dothiorella prunicola CBS 124723, lipases from Diplodia pinea (CAA 015 and CBS 109726), and proteases from Melanops tulasnei CBS 116806 were more active at 70 °C than at any of the other temperatures tested. In addition, lipases produced by Diplodia pinea were found to be significantly more active than any other known lipase from Botryosphaeriales. The thermal activity profile and the wide array of activities secreted by these fungi make them optimal producers of biotechnologically relevant enzymes that may be applied in the food and the health industries (proteases), the pulp-and-paper and biofuel industries (cellulases), or even in the detergent industry (lipases, proteases, amylases, and cellulases).

Botryosphaeriaceae and Diaporthaceae associated with panicle and shoot blight of pistachio in California, USA

Botryosphaeria panicle and shoot blight was considered as one of the single greatest threats to the California pistachio industry in the last two decades. A large number of fungi with typical morphological characteristics of Botryosphaeriaceae and Diaporthe were collected from pistachios in 18 counties in California and deposited in our culture collection. The aims of this study were to identify these isolates, recognize the distribution of these fungal species and test their pathogenicity to pistachio cultivars. A total of 304 California isolates were identified based on comparisons of DNA sequence data of the ITS, TEF-1α and β-tubulin gene regions, and combined with the morphological features of the cultures and conidia. Research results showed that California isolates represent eight species of Botryosphaeriaceae and one species of Diaporthe. These species include Botryosphaeria dothidea, Diplodia seriata, Dothiorella iberica, Dot. sarmentorum, Lasiodiplodia citricola, L. gilanensis, Neofusicoccum mediterraneum, Neof. vitifusiforme and Diaporthe chamaeropis. Of the Botryosphaeriaceae, 86 % of the isolates were identified as Neof. mediterraneum, which distributed in all the sampled counties. On pistachio trees, in addition to isolates from California, Neof. mediterraneum from Arizona, Neof. australe from Australia, B. dothidea, Neof. parvum and Dia. viticola from Greece were also identified. Pathogenicity of the California fungi on pistachio cultivars, Kerman (female) and Peters (male), using a mycelium plug and conidial suspension inoculation methods showed that all these species are pathogenic to the two tested pistachio cultivars, with L. citricola, L. gilanensis being the most pathogenic species, followed by Neof. mediterraneum. This study represents the first comprehensive work on the species identification, distribution and pathogenicity of Botryosphaeriaceae and Diaporthe on pistachio in California.

Postharvest Fruit rot of Persimmon (Diospyros kaki) in Spain Caused by Lasiodiplodia theobromae and Neofusicoccum spp.

AbstractThree different fungi (isolates IVIA QCV‐1, IVIA QCV‐3 and IVIA QCV‐4) were isolated as potential causal agents of postharvest decay losses observed on sweet persimmons (Diospyros kaki L.) cv. ‘Rojo Brillante’ from commercial packinghouses in the Valencia area (Spain). Disease symptoms were irregular brownish and soft lesions mainly located under and surrounding the fruit calyx (stem‐end) that expanded rapidly at room temperature and turned to dark brown or black colour producing apparent and in some cases abundant white to grey mycelium. The identification of Lasiodiplodia theobromae, Neofusicoccum mediterraneum and Neofusicoccum luteum by macroscopic and microscopic morphological observations was confirmed with the amplification and subsequent sequencing of the ribosomal DNA ITS2 region. Representative nucleotide sequences were deposited in GenBank. Pathogenicity of all three isolates was demonstrated by fulfilling Koch's postulates.

Olive Twig and Branch Dieback: Etiology, Incidence, and Distribution in California.

Eighteen different fungal species were isolated from symptomatic wood of olive trees (Olea europaea) affected by twig and branch dieback in California and identified by means of morphological characters and multigene sequence analyses of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequence of the β-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). These species included Diaporthe viticola, Diatrype oregonensis, Diatrype stigma, Diplodia mutila, Dothiorella iberica, Lasiodiplodia theobromae, Phaeomoniella chlamydospora, Phomopsis sp. group 1, Phomopsis sp. group 2, and Schizophyllum commune, which are for the first time reported to occur in olive trees; Eutypa lata, Neofusicoccum luteum, Neofusicoccum vitifusiforme, and Phaeoacremonium aleophilum, which are for the first time reported to occur in olive trees in the United States; and Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum mediterraneum, and Trametes versicolor, which have been previously reported in olive trees in California. Pathogenicity studies conducted in olive cultivars Manzanillo and Sevillano showed N. mediterraneum and Diplodia mutila to be the most virulent species and Diatrype stigma and D. oregonensis the least virulent when inoculated in olive branches. Intermediate virulence was shown for the rest of the taxa. This study demystifies the cause of olive twig and branch dieback and elucidates most of the fungal pathogens responsible for this disease in California.

Phylogeny and taxonomy of Botryosphaeria and Neofusicoccum species in Iran, with description of Botryosphaeria scharifii sp. nov.

Species of Botryosphaeriaceae are important pathogens and endophytes associated with woody plants. Botryosphaeria and Neofusicoccum are two well known genera of the family. In this study 125 isolates morphologically resembling members of this family were collected from about 20 different fruit and forest trees in Iran. Based on morphology, MSP-PCR profile and DNA sequence data (ITS and tef1-a), four species were identified. Of these, Botryosphaeria dothidea, Neofusicoccum mediterraneum and N. parvum are known while Botryosphaeria scharifii is described here as new. N. mediterraneum is a new record for Iran and is reported here for the first time on mango trees. High diversity within Iranian population of N. parvum suggests the need to revise and reassess the morphological species description of N. parvum and closely related species.

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

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.

First Report of Neofusicoccum mediterraneum and N. australe Causing Decay in Vitis vinifera in Castilla y León, Spain.

During a survey for grapevine decline, five young grapevines (cvs. Tempranillo and Viura) with low vigor and reduced foliage were collected (June and August 2009). Fungal isolations were performed from vascular and brown wood. Small pieces of brown wood were placed onto malt extract agar supplemented with 0.25 g/liter of chloramphenicol and incubated at 25°C in darkness. Five resulting colonies were transferred to potato dextrose agar (PDA). Isolates were characterized by abundant, gray, aerial mycelium that reached a radius of 45 mm after 4 days. Pycnidia induced on water agar with pine needles and UV light contained conidia that were hyaline, smooth, thin walled, fusiform, (20-) 22 to 26 (-28) × (5.5-) 6 (-6.5) μm, with granular cytoplasm. On the basis of morphological characteristics Neofusicoccum mediterraneum was suspected (1). Single-conidial cultures were generated from each isolate. DNA analyses were described in Martin and Cobos (2). Sequences of the internal transcribed spacer (ITS) region confirmed the identification and revealed 99% genetic identity with N. mediterraneum (GenBank Accession No EU040221). A sequence of the ITS fragment was deposited with Accession No. JF437919. Partial sequences of β-tubulin and 1-α elongation factor genes were amplified and deposited in the GenBank with Accession Nos. JF437921 and JF437923, showing 100 and 99% similarity to Accession Nos. GU292786 and GU251350, respectively. Pathogenicity tests were conducted with two isolates. The inoculations were carried out on a fresh wound on which an agar plug was applied; on 110R-rootstock woods of 12 young vines with N. mediterraneum and 12 other control plants were treated with agar only. Grapevines were maintained in a greenhouse at 20 to 25°C. After 4 months, N. mediterraneum was reisolated from vascular and brown tissues in 92% of inoculated plants, fulfilling Koch's postulates. Control plants were asymptomatic and N. mediterraneum was not recovered. With the same methodology, isolate Y264-21-1 reached a radius of 43 mm after 4 days at 25°C on PDA, presented colonies becoming olivaceous with a moderately dense mycelia, mat in center, and aerial around. Conidia were hyaline, fusiform, base subtruncate (19-) 23 to 26 (-31) × 5 to 6 (7.5) μm, unicellular, and smooth with granular contents. Based on these descriptions, N. australe was suspected (3). ITS sequence comparison revealed 99% genetic identity with N. australe (Accession No. FJ150697), a sequence of the fragment was deposited with Accession No. JF437920. Partial sequences of β-tubulin and 1α-elongation factor were deposited in the GenBank (Accession Nos. JF437922 and JF437924) showing 100 and 99% similarity to Accession Nos. AY615149 and GU251352, respectively. Koch's postulates were completed as described above. After 4 months, N. australe was reisolated from internal brown lesions in 92% of inoculated plants. Control plants were asymptomatic and N. australe was not recovered. The streaking length average from inoculation point for N. mediterraneum was 42 ± 22 mm and 53 ± 7 mm for N. australe. To our knowledge this is the first report of N. mediterraneum and N. australe in Castilla y León (Spain).

Characterization and Pathogenicity ofBotryosphaeriaceaeSpecies Collected from Olive and Other Hosts in Spain and California

Species in the family Botryosphaeriaceae are common pathogens causing fruit rot and dieback of many woody plants. In this study, 150 Botryosphaeriaceae isolates were collected from olive and other hosts in Spain and California. Representative isolates of each type were characterized based on morphological features and comparisons of DNA sequence data of three regions: internal transcribed spacer 5.8S, β-tubulin, and elongation factor. Three main species were identified as Neofusicoccum mediterraneum, causing dieback of branches of olive and pistachio; Diplodia seriata, causing decay of ripe fruit and dieback of olive branches; and Botryosphaeria dothidea, causing dalmatian disease on unripe olive fruit in Spain. Moreover, the sexual stage of this last species was also found attacking olive branches in California. In pathogenicity tests using unripe fruit and branches of olive, D. seriata isolates were the least aggressive on the fruit and branches while N. mediterraneum isolates were the most aggressive on both tissues. Isolates of B. dothidea which cause dalmatian disease on fruit were not pathogenic on branches and only weakly aggressive on fruit. These results, together with the close association between the presence of dalmatian disease symptoms and the wound created by the olive fly (Bactrocera oleae), suggest that the fly is essential for the initiation of the disease on fruit. Isolates recovered from dalmatian disease symptoms had an optimum of 26°C for mycelial growth and 30°C for conidial germination, suggesting that the pathogen is well adapted to high summer temperatures. In contrast, the range of water activity in the medium for growth of dalmatian isolates was 0.93 to 1 MPa, which was similar to that for the majority of fungi. This study resolved long-standing questions of identity and pathogenicity of species within the family Botryosphaeriaceae attacking olive trees in Spain and California.

First Report of Twig and Branch Dieback of English Walnut (Juglans regia) Caused by Neofusicoccum mediterraneum in California.

California produces 99% of the U.S. English walnut crop with more than 30 cultivars on ~89,000 ha. Production for 2008 was ~436,000 tons with a value of $527 million. In early summer of 2009 and 2010, branch and twig dieback of English walnut (Juglans regia L.) was detected in orchards in Yolo County and submitted to our diagnostic laboratory. Disease symptoms included death of twig tips, branch dieback, wood lesions, and canker formation. Pycnidia were embedded within the bark of dead twigs. Conidia from pycnidia were hyaline, fusoid-ellipsoidal, widest usually in the middle, and 21 to 24 (-27) × 5 to 7 μm (n = 30). Isolations from cankers yielded the fungus Neofusicoccum mediterraneum Crous, M.J. Wingf. & A.J.L. Phillips (1). Fungal colonies of N. mediterraneum grew light olive green to gray on potato dextrose agar, becoming dark olive green with age. Identification of fungal isolates was confirmed by sequence comparison of Californian isolates with ex-type (CBS 121558) sequences in GenBank (3) using the internal transcribed spacer region of the rDNA, a portion of the β-tubulin gene, and part of the translation elongation factor. Sequences of Californian isolates (GenBank HM443604-HM443609) were identical to the ex-type sequences for all three genes. Previous studies in California reported the occurrence and pathogenicity of N. mediterraneum into grapevine (Vitis vinifera L.) (3) and almond trees (Prunis dulcis L.) (2). Inderbitzin et al (2) investigated the host range of N. mediterraneum in California and reported the occurrence of pycnidia on English walnut trees. However, this study did not investigate the pathogenicity of N. mediterraneum on this host. In the current study, the pathogenicity of N. mediterraneum in J. regia cvs. Hartley and Chandler was investigated in an orchard at UC Davis using two fungal isolates. Pathogenicity tests were performed by inoculating eight 2- to 4-year-old branches of mature J. regia trees. Inoculations were made in June 2009 with a 5-mm cork borer to remove bark and placing an 8-day-old 5-mm-diameter agar plug bearing fresh mycelium of the fungal isolates directly into the fresh wound, mycelium side down. An additional eight branches of each cultivar were inoculated with noncolonized agar plugs to serve as controls. Inoculated wounds were covered with petroleum jelly and wrapped with Parafilm to retain moisture. Branches were harvested after 10 months of incubation and checked for canker development. The extent of vascular discoloration was measured in each branch and isolations were made from the edge of discolored tissue to confirm Koch's postulates. Statistical analyses were performed with analysis of variance and Dunnett's t-test to assess significant differences in the extent of vascular discoloration between inoculations with N. mediterraneum and the control. Necrosis length for the two isolates averaged 131.5 mm in Hartley branches and 110 mm in the Chandler branches. Average necrosis lengths in the control branches were 18.5 mm and 16.7 mm, respectively, significantly lower (P < 0.05) than the average necrosis length found in branches inoculated with N. mediterraneum. Fungal recovery was 75% in both varieties. To our knowledge, this study is the first report of N. mediterraneum as a pathogen of J. regia trees in California.

First Report of Grapevine Cankers Caused by Lasiodiplodia crassispora and Neofusicoccum mediterraneum in California.

Several species in the Botryosphaeriaceae family cause perennial cankers in the vascular tissue of grapevines and are responsible for the disease known as bot canker in California (3). Tissue from grapevine vascular cankers from samples submitted to our laboratory in the summer of 2009 were plated onto potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride. Lasiodiplodia crassispora (Burgess & Barber) and Neofusicoccum mediterraneum (Crous, M.J. Wingf. & A.J.L. Phillips) were identified based on morphological and cultural characters as well as analyses of nucleotide sequences. L. crassispora isolates were characterized by a fast-growing, white mycelium that turned dark olivaceous with age on PDA. Conidia from pycnidia formed in cultures were thick walled and pigmented with one septum and vertical striations when mature. Conidia measured (25.8-) 27.5 to 30.5 (-33.4) × (12.1) 14.3 to 16.8 (-18.2) μm (n = 60). Pycnidia contained septate paraphyses. N. mediterraneum was characterized as having moderately fast-growing, light green mycelia on PDA. Pycnidia formation was induced with pine needles placed on 2% water agar. Conidia from pycnidia were hyaline, ellipsoidal, thin walled, unicellular, and measured (18.2-) 20.5 to 27.8 (-29) × (5.1) 5.9 to 6.5 (-7.2) μm (n = 60). DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2), part of the β-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from L. crassispora (UCD23Co, UCD24Co, and UCD27Co) and N. mediterraneum (UCD695SJ, UCD719SJ, UCD720SJ, UCD749St, and UCD796St) grapevine isolates from California were amplified and sequenced. Consensus sequences from L. crassispora and N. mediterraneum from California showed 99 to 100% homology with L. crassispora and N. mediterraneum isolates previously identified and deposited in GenBank (1,2). Sequences from the examined DNA regions of all isolates were deposited at GenBank (GU799450 to GU799457 and GU799473 to GU799488). Pathogenicity tests using three isolates per species were conducted on detached dormant canes of cv. Red Globe. Ten canes per isolate were inoculated by placing a 7-day-old 5-mm-diameter agar plug from each fungal culture into a wound made with a drill on the internode (4). Twenty shoots were inoculated with noncolonized PDA plugs for negative controls. Six weeks after inoculations, necrosis was measured from the point of inoculation in both directions. One-way analysis of variance was performed to assess differences in the extent of vascular discoloration and means were compared using Tukey's test. L. crassispora isolates caused an average necrotic length of 21.1 mm, which was significantly lower (P < 0.05) than the average necrotic length of 35.6 mm caused by the N. mediterraneum isolates. Reisolation of L. crassispora and N. mediterraneum from necrotic tissue was 100% for each species. The extent of vascular discoloration in infected canes was significantly greater (P < 0.05) than in control inoculations (8 mm) from which no fungi were reisolated from the slightly discolored tissue. To our knowledge, this is the first report of L. crassispora and N. mediterraneum as pathogens of Vitis vinifera and as a cause of grapevine cankers in California.