Characterisation and pathogenicity of Pestalotiopsis uvicola and Pestalotiopsis clavispora causing grey leaf spot of mango (Mangifera indica L.) in Italy

During 2009 and 2010, twenty-one isolates of Pestalotiopsis spp. Associated with grey patches on the leaves, twigs, and panicles of mango were collected in six orchards located in Sicily (Italy). Morphological characteristics of colony (colour and mycelium appearance), and conidia (size, shape, septation, length and the number of apical and the basal appendages) as well as phylogenetic analysis of the Internal Transcribed Spacer (ITS) region (ITS1, 5.8S gene, and ITS2) of six representative isolates revealed the occurrence of P. uvicola and P. clavispora. The representative isolates of both species were pathogenic to the artificially inoculated detached mango leaves cv. Kensington Pride and showed significant variation in lesion size. This is the first report worldwide of P. uvicola and P. clavispora causing grey leaf spot of mango.     

Characterisation of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> isolates from <Emphasis Type="Italic">Vitis vinifera</Emphasis> and potential biocontrol activity for the management of bitter rot of grapes

Aureobasidium isolated from Vitis vinifera (cv Chardonnay) grapevine tissues were characterised using morphological and molecular techniques. Species level identification of 29 isolates was accomplished by partial amplification and sequencing of the ITS region (ITS1–5.8S–ITS2) using universal primers ITS1 and ITS4. A comparison of nucleotide sequences using BLAST followed by phylogenetic analysis revealed that all isolates examined were Aureobasidium pullulans. Strain level discrimination of a total of 100 epiphytic Aureobasidium isolates including three reference strains was successfully carried out using two inter simple sequence repeat (ISSR) primers, (AAC)5 and (GTG)5 and the Intron Splice Junction R1 (ISJ-R1) primer in which 24, 24 and 15 scorable bands were produced for each primer, respectively. The high level of genetic variation recorded among the isolates further highlighted the high levels of strain diversity among A. pullulans residing on grapevines. Thirty-two epiphytic Aureobasidium isolates were examined for their ability to inhibit the growth of Greeneria uvicola, responsible for bitter rot of grapes. Using an in-vitro dual-culture antagonism assay, all isolates inhibited the growth of G. uvicola (Isolates DAR 77272 and DAR 77273) with inhibition ranging from 15 to 85%. Three Aureobasidium isolates were then examined for their ability to inhibit G. uvicola when co-inoculated onto detached berries, leaves and grape bunches growing on potted vines in a glass house. All isolates reduced the severity of bitter rot infection. The results indicate that A. pullulans has the potential to suppress bitter rot of grapes.     

四株虫生真菌的鉴定及其对小菜蛾的室内毒力

为了明确4株虫生真菌在小菜蛾防治中的应用潜力,采用生物测定法测定其室内毒力,并通过多相分类法研究其分类地位。结果表明:菌株GZU-BCECWC113对小菜蛾的毒力最强,在1.6×108孢子/mL浓度下对小菜蛾的校正死亡率最高,达到89.47%;半致死时间LT50值最小,为3.33天;感染8天时的LC50值最小,为0.45×105孢子/mL。根据形态特征、培养性状及ITS序列分析结果明确4株菌的分类地位,即GZU-BCECWC113为环链拟青霉Paecilomyces catenianulatus,GZU-BCECWQ16为玫烟色拟青霉P.fumosorosenus,GZU-BCECW122为蝉拟青霉P.cicadae,GZU-BCEC-WKKS2为细脚拟青霉P.tenuipes。     

First report of bacterial blight of crucifers caused by Pseudomonas cannabina pv. alisalensis in Japan

In October 2010, a bacterial disease produced flecks and spots on leaves of Chinese cabbage, cabbage and Japanese radish in Nagano Prefecture, Japan. The symptoms started on the abaxial surface of leaves as angular, water-soaked flecks of 1–2 mm in diameter with a yellow halo of 3–4 mm width. These flecks then became visible on both leaf surfaces, enlarged and coalesced into large blight lesions. The symptoms were similar to bacterial leaf spot caused by Pseudomonas syringae pv. maculicola. The bacterium isolated from leaf lesions formed a white colony and produced polysaccharides on YP agar. The isolates were identified as P. syringae group by LOPAT tests and the 16S rDNA sequence. Moreover, the results of pathogenicity on cruciferous plants, bacteriological characteristics, rep-PCR and the sequences of rpoD and gyrB showed that the isolates should be identified as P. cannabina pv. alisalensis (recently transferred from P. syringae pv. alisalensis). This is the first report of P. cannabina pv. alisalensis isolated from diseased crucifers in Japan.     

Bacterial brown spot on Avena storigosa Schereb. caused by Pseudomonas syringae pv. alisalensis

Avena storigosa Schereb. (bristle oat) is used as a green manure in crop rotations and as an antagonist of nematodes in Nagano Prefecture, Japan. In 2011, necrotic, brown, water-soaked lesions were observed on young bristle oat plants. A pathogenic bacterium was isolated from symptomatic leaves of infected plants and produced the same symptoms after inoculation. Bacteriological properties of the bacterial isolates from bristle oat matched those of Pseudomonas syringae pathovars. The host range of the bristle oat isolates was identical to that of P. syringae pv. alisalensis. This is the first report of bristle oat disease caused by P. syringae pv. alisalensis.     

Characterization of three Colletotrichum acutatum isolates from Capsicum spp.

Colletotrichum acutatum causes anthracnose on peppers (Capsicum spp.), resulting in severe yield losses in Taiwan. Fungal isolates Coll-153, Coll-365 and Coll-524 collected from diseased peppers were found to differ in pathogenicity. Pathogenicity assays on various index plants revealed that Coll-524 was highly virulent and Coll-153 was moderately virulent to three commercially available pepper cultivars. Both isolates induced anthracnose lesions and produced abundant conidia. Coll-365 was only weakly virulent on pepper fruit, where it caused small lesions and hardly produced conidia on pepper fruit. However, Coll-365 was highly pathogenic to tomato fruit and mango leaves, where it caused anthracnose lesions and formed acervuli and conidia. All three isolates showed similar abilities in the attachment and germination of conidia, formation of highly branched hyphae and appressoria, penetration of cuticles, and infection of epidermal cells on chili peppers. Coll-365 accumulated less turgor pressure in appressoria but produced higher levels of cutinase and protease activity than Coll-153 and Coll-524 did. All three isolates invaded the neighbouring cells through plasmodesmata in chili peppers and showed similar pectinase or cellulase activities in culture. However, the most virulent strain Coll-524 expressed stronger laccase activity and was more resistant to capsaicin compared to Coll-153 and Coll-365. The three isolates are different in numbers and sizes of double-stranded RNAs. Depending on the cultivar genotypes, cellular resistance of chili pepper to C. acutatum might rely on the ability to restrict penetration, colonization, or conidiation of the pathogen. We conclude that the differences in pathogenicity among the three C. acutatum isolates of pepper are attributed to their ability to colonize the host plant.     

A pith necrosis caused by Xanthomonas perforans on tomato plants

Pith necrosis is a common disease of tomato in Europe, mainly caused by Pseudomonas corrugata and other soil-borne species of Pseudomonas. During 2011–2012 a survey was conducted in soil-grown tomato crops in southeastern Sicily (Italy). Plants showed pith necrosis, brown discolouration of the vascular tissues, leaf chlorosis and sometimes wilting of leaves. Thirty bacterial isolates from symptomatic tissues, forming colonies on NA and KB, were identified by morphological, biochemical and physiological tests. Among them, seven isolates were analyzed for their 16S rDNA and 16S–23S spacer region sequence that resulted in 99 % identity to that of the Xanthomonas perforans type strain (GenBank accession number GQ46173over 2.085 bp.). Additional sequences of fusA, gapA, gltA, gyrB, lacF, and lepA from one selected isolate were 100% identical to sequences of the Xanthomonas perforans type strain. X. perforans local isolates showed similar genomic patterns with REP-PCR and fAFLP, and were clearly distinguished from other Xanthomonas spp. type strains. In stem-inoculation assays, bacteria isolated from symptomatic tomato plants identified as P. fluorescens, P. putida, P. marginalis, P. citronellolis, P. straminea, and Pantoea agglomerans induced discolouration of vascular tissues, while Pectobacterium carotovorum subsp. atrosepticum isolates induced soft rot. Conversely, the isolates here identified as Xanthomonas perforans were able to induce pith necrosis, vascular discolouration, longitudinal splits and external lesions on stems. This report of X. perforans causing pith necrosis on tomato represents a potentially serious problem that may limit the productivity of tomato crops.     

Ilyonectria palmarum sp. nov. causing dry basal stem rot of Arecaceae

During surveys conducted in 2010–2013, a complete breakage or bending of the trunk and a dry basal stem rot were observed on containerised Brahea armata, B. edulis, Howea forsteriana and Trachycarpus princeps plants in different nurseries located in eastern Sicily (southern Italy). A cylindrocarpon-like species was consistently obtained from diseased palm tissues, while known pathogens of these hosts such as Ganoderma, Phytophthora and Thielaviopsis were not found associated with symptomatic tissues or isolated on standard or selective media. A total of 40 cylindrocarpon-like isolates were collected and characterised based on morphology and DNA phylogeny. Multigene analyses based on the β-tubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers (ITS1, 5.8S, ITS2) genes facilitated the identification of a new species, described here as Ilyonectria palmarum. The pathogenicity of one representative isolate collected from each palm species was tested on plants cultivated under nursery conditions and in a growth chamber. All isolates were pathogenic to B. armata, B. edulis, H. forsteriana, and T. princeps and symptoms identical to that observed in nurseries were reproduced. Dry basal stem rot and stem bending caused by Ilyonectria palmarum represents a potentially serious problem for nurseries cultivating containerised palms.     

Alternaria Leaf Spot on Mealycup Sage (<Emphasis Type="Italic">Salvia farinacea </Emphasis>Benth.) Caused by <Emphasis Type="Italic">Alternaria alternata </Emphasis>(Fr.) Keissler

In June 1995, a disease causing round to irregular-shaped, water-soaked, brown to blackish brown spots on mealycup sage (Salvia farinacea Benth.) was found in Atsugi-shi, Kanagawa Prefecture, Japan. The symptoms were seen only on leaves, not on neither flower petals or stems. The disease was also found in Setagaya-ku, Tokyo, Memambetsu-cho, Hokkaido and Shimoda-shi and Matsuzaki-cho, Shizuoka. An Alternaria sp. was frequently isolated from these diseased plants. The isolates were severely pathogenic to mealycup sage and caused lesions on the inoculated leaves. The isolates were also weakly pathogenic on scarlet sage (S. splendens Sellow ex Roem. and Schult.) but not on any other Labiatae plants tested. Based on morphological characteristics, such as size of conidia, chain number, and the short beak on conidia, the causal fungus was identified as Alternaria alternata (Fr.) Keissler. This report is the first on a mealycup sage disease caused by A. alternata. Because the symptom was restricted to the leaf, the common name of Alternaria leaf spot was proposed. Received 30 August 2002/ Accepted in revised form 18 November 2002     

First report of downy mildew of carnation caused by Peronospora dianthicola in Japan

Yellowish lesions with downy, gray growth developed on the leaves of carnations (Dianthus caryophyllus) in a greenhouse in Hokkaido Prefecture, Japan, in March 2011. We identified the fungus on diseased plants as Peronospora dianthicola on the basis of the morphologies of the conidia, conidiophores, and oospores. To confirm pathogenicity of the fungus, we inoculated healthy plants with a conidial suspension of the fungus, and the plants developed the same signs and symptoms as the naturally infected plants. We then reidentified P. dianthicola from the diseased lesions. This is the first report of downy mildew of carnation in Japan.     

First report of leaf spot disease on oil palm caused by Pestalotiopsis theae in Thailand

A leaf spot on oil palm, caused by Pestalotiopsis theae, was found in a plantation of Elaeis guineensis for the first time in the world in Chiang Mai Province, Thailand. The fungus was isolated from lesions on leaves, and its pathogenicity was confirmed. Pathogenicity tests showed that P. theae could infect E. guineensis, which developed the same symptoms after inoculation as those observed naturally in the field. The fungus was identified based on morphological characteristics and confirmed using comparisons of DNA sequences of internal transcribed spacer (ITS)1, ITS2 and 5.8S rDNA. This report is the first on oil palm leaf spot disease caused by P. theae.     

Characterization and epidemiology of Colletotrichum acutatum sensu lato (C. chrysanthemi) causing Carthamus tinctorius anthracnose

In 2012, Colletotrichum isolates were collected from field‐grown safflower (Carthamus tinctorius) crops in central Italy from plants exhibiting typical anthracnose symptoms. Colletotrichum isolates were also collected from seed surfaces and from within seeds. The genetic variability of these isolates was assessed by a multilocus sequencing approach and compared with those from Colletotrichum chrysanthemi and Colletotrichum carthami isolates from different geographic areas and other Colletotrichum acutatum sensu lato‐related isolates. Phylogenetic analysis revealed that all of the strains isolated from C. tinctorius belonged to the species described as C. chrysanthemi, whereas all of the strains belonging to C. carthami had been isolated from Calendula officinalis. Phenotypic characterization of isolates was performed by assessing growth rates at different temperatures, morphology of colonies on potato dextrose agar (PDA) and the size of conidia. All C. chrysanthemi isolates from safflower had similar growth rates at different temperatures, comparable colony morphologies when grown on PDA and conidial sizes consistent with previously described C. chrysanthemi isolates. Pathogenicity tests were performed by artificially inoculating both seeds and plants and confirmed the seedborne nature of this pathogen. When inoculated on plants, C. chrysanthemi caused the typical symptoms of anthracnose on leaves. This is the first record of this pathogen on C. tinctorius in Italy, and it presents an updated characterization of Colletotrichum isolates pathogenic to safflowers in Europe.     

Biological characterization and genetic diversity analysis of two species of Pestalotiopsis causing twig dieback of Myrica rubra

Chinese bayberry (Myrica rubra Siebold & Zucc) is an evergreen fruit tree with high ecological and economic values in China. In recent years, a new twig dieback disease caused by Pestalotiopsis mangiferae and P. vismiae was observed in major M. rubra-producing areas of Zhejiang and Fujian Provinces, causing serious economic losses. In this study, 16 isolates of P. mangiferae and 27 isolates of P. vismiae were obtained from diseased leaves, roots and branches of M. rubra from different regions. The optimum growth temperature of the two species of Pestalotiopsis was determined to be 20–25 °C, while the optimum temperature for the germination of conidia was 25–35 °C. The two species of Pestalotiopsis showed rich genetic diversity. Inoculating the conidial suspension of one or both of the two species of the Pestalotiopsis on detached leaves or branches of M. rubra could cause lesions surrounding the inoculation sites with the frequency of 100 %. Moreover, necrotic lesions could be observed on inoculated potted plants with the frequencies of 33.3 % for P. mangiferae, 25 % for P. vismiae and 50 % for a mixed inoculum.     

Occurrence and characterisation of <Emphasis Type="Italic">Rhizoctonia</Emphasis> species causing diseases of ornamental plants in Italy

During surveys conducted in 2010–2012 Rhizoctonia symptoms were observed on 30 ornamental species in different nurseries located in eastern Sicily (Southern Italy). Eighty-eight isolates of Rhizoctonia spp. were obtained from symptomatic leaves, roots and stems. Fifty-six of the isolates were binucleate and 32 were multinucleate Rhizoctonia. Characterisation of anastomosis groups (AGs) was performed using morphological characteristics and sequence analysis of the internal transcribed spacer of ribosomal DNA ( rDNA-ITS) region. Most isolates collected were Rhizoctonia solani AG-4 HG-I (35.2% of all isolates) and one isolate was AG-2-2 IIIB. The binucleate isolates belonged to AG-R (27.3%), AG-A (21.6%), AG-G (12.5%), AG-V (1.1%) and AG-Fb (1.1%). The pathogenicity of 38 representative isolates collected from each host was tested on seedlings or cuttings grown in a growth chamber. All R. solani AG-4 HG-I isolates, most of the binucleate AG-R, AG-A and AG-G and AG-V were pathogenic and reproduced symptoms identical to that observed in nurseries, while binucleate AG-Fb and R. solani AG-2-2 IIIB isolates were nonpathogenic. This is the first report of the occurrence of Rhizoctonia species on some ornamental plants and the first report of binucleate Rhizoctonia AG-R and AG-V in Europe.     

Valdensia leaf blight of highbush blueberry caused by Valdensinia heterodoxa, a new fungal disease in Japan

In July 2003, a new disease occurred on leaves of highbush blueberry (Vaccinium corymbossum L.) in Iwate, Japan. Leaves initially had brownish spots, which subsequently developed into large lesions with concentric rings, resulting in premature defoliation. Teardrop-shaped conidia infecting leaves were visible by the naked eye as small protuberances in the center of lesions. Star-shaped conidia were sporadically produced on large lesions. The causal fungus was identified as Valdensinia heterodoxa, based on cultural, morphological and genetic studies. Inoculation tests showed that the fungus reproduced lesions on detached young leaves of highbush blueberry. A field survey in 2009 indicated that symptoms initially appeared on the lower leaves of basal shoots in late May, and the disease rapidly progressed on leaves of basal shoots, eventually spreading to lateral shoots from late June to late July. Removal of all basal shoots in late June significantly reduced disease incidence on lateral shoots in late July.     

苹果炭疽叶枯病病原Glomerella cingulata及其侵染过程

为明确苹果炭疽叶枯病病原菌围小丛壳Glomerella cingulata的侵染致病特征,在分离获得该病原菌的基础上,采用形态学观察、ITS序列分析和致病性测定对其进行了鉴定,并利用光学和扫描电子显微镜对病原菌在嘎啦苹果叶片上的侵染过程进行了研究.结果表明,在陕西咸阳地区分离获得的9株病原菌均为围小丛壳G.cingulata.25 ℃下接种9 h后,分生孢子中间产生隔膜,双胞化,并萌发产生芽管和附着胞;24 h后分生孢子的2个细胞均可萌发并形成芽管及附着胞,部分芽管顶端可产生次级分生孢子;48 h后次级分生孢子萌发形成附着胞;72 h后,附着胞下形成的侵染钉可直接入侵寄主,在表皮细胞内形成初生菌丝和次生菌丝,此时叶片表面已出现褐色斑点.接种7 d后叶片病斑处出现分生孢子盘和子囊壳.表明陕西省近年出现的苹果炭疽叶枯病病原菌为围小丛壳G.cingulata,该病菌在嘎啦叶片上的一些特殊侵染行为可能是导致该病害易在短时间内暴发的重要原因.     

Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

Phylogenetic relationships,pathogenicity and fungicide sensitivity of Greeneria uvicola isolates from Vitis vinifera and Muscadinia rotundifolia grapevines

Greeneria uvicola causes bitter rot on Vitis vinifera (bunch grapes) and Muscadinia rotundifolia (muscadine grapes) in warm moist temperate and subtropical regions. This study investigated the phylogenetic relationship of G. uvicola representatives from Australia (67 isolates), the USA (31 isolates), India (1 isolate) and Costa Rica (1 isolate) and compared their pathogenicity and fungicide sensitivity. Differences in cultural and conidial morphology were observed between the isolates from Australia and the USA. Phylogenetic relationships were determined based on three gene regions: the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1–5?8S–ITS2), 28S large subunit (LSU) nuclear rDNA and β‐tubulin‐2. Greeneria uvicola isolates were clearly differentiated into four groups: isolates from Australia and India; USA isolates from V. vinifera; USA isolates from M. rotundifolia; and the isolate from Costa Rica. All isolates were pathogenic on V. vinifera (cv. Chardonnay) berries although those originating from M. rotundifolia were not as aggressive as isolates from V. vinifera, irrespective of geographical origin. Sensitivity to pyraclostrobin and salicylhydroxamic acid (SHAM) was studied. Despite differences in fungicide applications, hyphal growth inhibition was not significantly different for geographical location, cultivar, tissue, year of collection or different spray regimes. For the Australian and USA isolates, fungal growth inhibition was significantly greater for pyraclostrobin than for SHAM, and was significantly greater for the combined treatment than for each of the fungicides applied singly. The aetiological and epidemiological knowledge of bitter rot collected through this study will aid better prediction and management strategies of this pathogen.     

Characterization of Pectobacterium carotovorum subsp. odoriferum causing soft rot of stored vegetables

Pectobacterium carotovorum subsp. odoriferum has been generally considered to have a narrow host range and has been isolated most often from chicory. Research was conducted to identify 91 Pectobacterium spp. strains isolated from different vegetables in Europe, North and South America, Asia, and Africa, and to compare their ability to cause disease in chicory and potato. Among the 91 strains, 22 strains from Europe were identified as P. c. subsp. odoriferum. Based on phylogenetic analysis of 16S rDNA, recA, and rpoS gene sequences, strains isolated from stored vegetables clustered together with the type strain of P. c. subsp. odoriferum and clustered separately from the P. c. subsp. carotovorum isolates. Eleven strains previously identified as P. c. subsp. carotovorum were reclassified as P. c. subsp. odoriferum. All P. c. subsp. odoriferum isolates were able to cause soft rot symptoms on chicory and potato. Moreover, the symptoms on potatoes were more severe at temperatures from 15 to 37 °C with P. c. subsp. odoriferum isolates than with P. atrosepticum or P. c. subsp. carotovorum isolates. Tissue maceration by P. c. subsp. odoriferum isolates was highest at 28 °C, and at that temperature tissue maceration was two-times greater for P. c. subsp. odoriferum isolates than for P. c. subsp. carotovorum isolates. Symptoms on inoculated chicory leaves were more severe with P. c. subsp. odoriferum (regardless of origin) than with other subspecies or species. To our knowledge, this is the first report that P. c. subsp. odoriferum occurs on a wide range of vegetables and has the ability to cause soft rot during potato storage.     

Effect of inoculum composition on infection of French bean leaves by conidia of Botrytis cinerea

Inoculation of leaves of French bean (Phaseolus vulgaris) with sprays or small drops of a suspension of conidia ofBotrytis cinerea gave rise to spreading lesions, lesions remaining restricted in size or to no visible necrosis. The type of reaction depended on the composition of the inoculum. In studies with drop inoculations with buffered inocula some of the factors involved were analyzed. The formation of spreading lesions depended on pH, type and molarity of the buffer, presence of glucose, and concentration of conidia in the inoculum. If the phosphate buffer used in most of the inocula was replaced by monobasic phosphate, similar results were obtained. The reactions were not influenced by the proportion of K⁺ or Na⁺ ions in the phosphate buffer. Inoculations with conidia suspended in a solution of 0.067 M phosphate buffer (pH 5.0) or monobasic phosphate and 0.11 M glucose always evoked a susceptible reaction, i. e. the formation of spreading lesions.