Aleophilum Isolates Research Articles

Pleurostomophora richardsiae associated with trunk diseases of grapevines in southern Italy

Pleurostomophora richardsiae (Nannf. apud Melin & Nannf.) L. Mostert, W. Gams & Crous was previously known mainly as a human pathogen. However, more recently this fungus has been isolated from wood tissue of grapevines that show Petri and esca disease symptoms in California (USA) and South Africa. During an assessment carried out in southern Italy, the abundant presence of this fungus was demonstrated by morphological, cultural and molecular means. Pleurostomophora richardsiae was isolated from sub-cortical wood patches and streaking of trunks and cordons of grapevine cultivars that showed decline and dieback symptoms. To understand its putative pathogenic role, pathogenicity tests were conducted in greenhouse experiments, where young grapevine plants of two cultivars were artificially inoculated with two isolates each of Pl. richardsiae, Lasiodiplodia theobromae and Phaeoacremonium aleophilum . Within 130 d, all three fungi produced brown streaking in both grapevine cultivars. The L. theobromae and Pl. richardsiae isolates were the most aggressive. Although the Pm. aleophilum isolates were pathogenic, they induced less severe wood streaking than the other two fungi. Therefore, Pl. richardsiae is considered a fungal pathogen of grapevine. All three fungal species were re-isolated from discolored tissue of all inoculated shoots, thus fulfilling Koch’s postulates.

Pome and stone fruit trees as possible reservoir hosts for Phaeoacremonium spp., the causal agents of grapevine esca disease, in Iran

Phaeoacremonium spp. are known as the main Hyphomycetes associated with esca disease symptoms on grapevines worldwide. The role of other woody hosts in proximity of vineyards as a possible inoculum source for the causal agents of grapevine esca disease mainly remains unknown. The aim of this study was to investigate the occurrence of Phaeoacremonium spp. on pome and stone fruit with trunk diseases symptoms in northwestern parts of Iran. For this purpose, wood trees samples were collected from apple and apricot trees with trunk disease symptoms. Isolation was made using routine plant pathology methods. Based on the morphological and cultural characteristics, Phaeoacremonium aleophilum and Phaeoacremonium iranianum were identified from apple trees and P. aleophilum was recovered from apricot trees with trunk disease symptoms. A phylogenetic analysis based on the sequence data from β-tubulin gene further confirmed the identity of the species as P. aleophilum and P. iranianum. The results of inoculation assay on detached shoots of apples revealed that P. iranianum is pathogenic on apple. Our results also showed that P. aleophilum isolates from different hosts (apple, apricot and grapevine) were pathogenic on apple and induced wood discolouration on apple stems. There was no significant difference on the length of lesions induced in relation to the origin of the P. aleophilum isolates. This study is the first report on the occurrence of P. iranianum on apple trees with trunk disease symptoms worldwide and the first report on the occurrence of P. aleophilum on apple and apricot in Iran.

Multi-gene sequence analysis and phenotypic diversity of Phaeoacremonium species isolated from grapevines in Spain

Esca and Petri disease, caused by Phaeoa-cremonium, and other grapevine decline diseases, such as eutypa dieback, are responsible for serious economic losses to the wine industry worldwide. In the present work, 175 isolates of Phaeoacremonium spp. were collected (mostly in the region of Castilla y Leon, Spain) from vines either asymptomatic or symptomatic for grapevine decline diseases. The sequences of DNA fragments of 30 isolates were analysed. Amplicons of ITS, and partial gene of the β-tubulin, actin, calmodulin and translation elongation factor 1-α [EF1-α], were obtained by PCR. This is the first time that any exon of the EF1-α gene or exons 6–7 of the β-tubulin gene of seven Phaeoacremonium spp. have been amplified. Amplicons of the calmodulin and EF1-α exons were the most informative in terms of species differentiation: only 52.6 % and 56.6 % similarity across six and seven species was respectively seen. Phylogenetic analysis of combined DNA sequences revealed five Phaeoacremonium species isolated from the sampled plants; P. aleophilum was the most common. Four other species were found, however only one isolate each. P. mortoniae and P. iranianum are here reported for the first time on grapevines in the region of Castilla y Leon and the neighbouring region of La Rioja respectively. In total, 29 nucleotide differences among the studied gene fragments were seen across 23 isolates of P. aleophilum. Network analysis showed these Spanish P. aleophilum isolates to fall into two main groups of genotypes. P. aleophilum genotypes belonging to group 1 were mostly isolated from young grapevines affected by Petri disease but genotypes included in group 2 were isolated from adult plants showing esca or eutypa dieback symptoms.

Characterization of fungicide resistant isolates of Phaeoacremonium aleophilum infecting grapevines in Spain

Fungicide resistance in Phaeoacremonium aleophilum, one of the most frequent fungal pathogens associated with grapevine trunk diseases, was investigated and found to exist in some isolates of the pathogen against a commercial formulation, Escudo®. The effect of this compound and its two active substances, carbendazim and flusilazole, was first evaluated on the mycelial growth of P. aleophilum. Escudo®-resistant isolates were estimated at a frequency of 24% in Spanish vineyards. Then, the two active substances were used individually to test their effect on mycelia growth of twelve single-spore cultures originating from six Escudo®-resistant isolates. Flusilazole (DMI-triazole) did not inhibit mycelia growth of any single-spore cultures of P. aleophilum. Carbendazim (benzimidazole) used alone allowed the growth of the same single-spore cultures that were also resistant to Escudo®. AFLP characterization of sensitive and resistant single-spore cultures showed genetic diversity within P. aleophilum isolates but no AFLP markers were associated with resistance. New primers set (L2/R1) were designed to partially amplify the exon 6 of the beta-tubulin gene of P. aleophilum. Two different point mutations resulted in glycine (GGC) or lysine (AAA) replacing the glutamic acid (GAG) at codon 198 of the beta-tubulin gene in some of the resistant single-spore cultures studied. Resistant single-spore cultures of P. aleophilum were shown to have different aggressiveness levels as sensitive single-spore cultures by inoculation of wood segments of Vitis vinifera in the presence and absence of fungicide.

Molecular and physiological diversity among Verticillium fungicola var. fungicola

The genetic and physiological variability of Verticillium fungicola var. aleophilum responsible for Agaricus bisporus dry bubble disease in North America is well documented but little is known about the var. fungicola affecting European crops. Variability was assessed within this variety and compared with that reported for the var. aleophilum. Eighteen isolates of V. fungicola var. fungicola and four var. aleophilum isolates were analysed for DNA polymorphism, mycelial growth, response to biochemicals produced by A. bisporus, fungicide resistance, and pathogenicity assessed by direct inoculation on sporophore or casing contamination. RAPD and AFLP markers delineated three French isolates from a homogeneous group containing the other var. fungicola isolates, but no correlation could be drawn between DNA polymorphism and the various traits studied. The var. fungicola isolates were more susceptible than the var. aleophilum isolates to the antibiosis effect of A. bisporus. Only mycelial growth rate at 23 °C could explain the variability in aggressiveness among the European isolates. The putative effect of the post-incubation temperature on contamination during mushroom cultivation was discussed. This work emphasized that, like the American var. aleophilum, the var. fungicola in Europe is genetically homogeneous, but physiological diversity exists, especially in France where it could be related to less standardized cultural practices.

Teleomorph Formation of Phaeoacremonium aleophilum, Cause of Esca and Grapevine Decline in California.

Phaeoacremonium is a recently described genus of the hyphomycetes and includes species associated with grapevine (Vitis vinifera) declines worldwide. Spores of Phaeoacremonium spp. have been trapped in infested vineyards, but neither asexual nor sexual fruiting structures have been observed in the field. Mating studies were carried out to determine if California P. aleophilum isolates are capable of forming a teleomorph in vitro. Sterilized grapevine shavings were placed on the surface of water agar plates with pairs of different California isolates of P. aleophilum, an isolate from the holotype of P. aleophilum, plus other related Phaeoacremonium spp. After approximately 28 to 35 days, perithecia were seen forming on wood chips and agar of many pairings. Upon maturation, fertile perithecia had gelatinous droplets of ascospores oozing from their ostioles. Successful crosses, resulting in mature perithecia, corresponded to a heterothallic mating type system. When F1 progeny were backcrossed with their parents, heterothallism was confirmed. Molecular analyses of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA from ascospore progeny and perithecia confirmed that these perithecia were the teleomorph of P. aleophilum, Togninia minima. Furthermore, 4 months after moist incubating grapevine pieces from naturally infected vineyards, mature perithecia of T. minima could be seen forming on the xylem and pith tissues, suggesting both mating types occur on the same vine.

Detection of the "Togninia" Teleomorph of "Phaeoacremonium aleophilum" in Australia

Moist incubation of grapevine wood infected with both Phaeoacremonium aleophilum and Phaeomoniella chlamydospora yielded an ascomycete referable to the genus Togninia (Ascomycota, Calosphaeriales). Single ascospore cultures were morphologically identical to Pm. aleophilum. The rDNA ITS sequence of single ascospore isolates was identical to published sequences for the majority of Pm. aleophilum isolates. Comparison with the morphology of other wood staining Togninia species confirms that the teleomorph of Pm. aleophilum is Togninia minima.

Genetic and physiological variation in isolates of Verticillium fungicola causing dry bubble disease of the cultivated button mushroom, Agaricus bisporus

The objectives of this study were to examine genetic variation, physiological dissimilarities and diversity in pathogenicity between Verticillium fungicola var. aleophilum and var. fungicola and within a population of six V. fungicola var. fungicola strains responsible for dry bubble outbreaks on mushroom farms. Genetic variability was investigated using random amplification of polymorphic DNA (RAPD). Mycelial growth rate, extracellular enzyme production and susceptibility to hydrogen peroxide were used to examine physiological dissimilarities. Variation in pathogenicity was studied both in vitro and during mushroom cultivation. All the physiological properties studied indicated that var. aleophilum isolates were potentially more efficient than var. fungicola isolates for rapid colonisation of the mushroom cultivation medium. They could then interact more efficiently with Agaricus bisporus to produce dry bubble disease. RAPD analysis confirmed that all the French isolates belonged to var. fungicola, and two isolates were distinguishable from the homogeneous group constituted by the others. These isolates had a higher mycelial growth rate and lower extracellular enzyme activities in liquid media, except for chitinases. Their spores were more susceptible to germination inhibition by hydrogen peroxide, and they were responsible for higher levels of affected mushrooms. The two varieties might be regarded as pathotypes that are geographically isolated, and variation in isolates of var. fungicola might have consequence for mushroom growers.

Partial ribosomal DNA sequences show an important divergence between Phaeoacremonium species isolated from Vitis vinifera

A morphological and molecular analysis was performed on 25 individuals of Phaeoacremonium isolated from diseased European and Californian grapevines (Esca disease). An unexpectedly high intra-generic molecular divergence was evidenced by partial rDNA sequencing between P. chlamydosporum isolates on one hand and an aggregate including P. angustius and P. aleophilum isolates and some undescribed ones on the other. These two groups show 26% nucleotide differences within the 26S rDNA fragment sequenced and were unalignable within the ITS2, suggesting distant phylogenetic relationships. Moreover, P. chlamydosporum appeared more closely related to Phialophora verrucosa (supposed to be an anamorph of the Herpotrichiellacae), than to the type species of the genus, P. parasiticum (more related to Magnaporthaceae).