Characterization of hypovirulent isolates of the chestnut blight fungus, Cryphonectria parasitica from the Marmara and Black Sea regions of Turkey

Chestnut blight caused by the introduced fungus Cryphonectria parasitica has been responsible for the decline of Castanea sativa in Turkey since the 1960s. In this study, 72 C. parasitica isolates were recovered from the Marmara and Black Sea regions of Turkey showing white or cream-coloured culture morphology and were subjected to various tests to determine if they were infected by Cryphonectria hypovirus 1 (CHV-1). The vast majority of the isolates (69 out of 72) were vc type EU-1. Both mating types were found among a subsample of the isolates. The hypovirus was detected in 55 isolates by dsRNA extraction and/or virus specific RT-PCR on total RNA extracts. All but one isolates showed no or only weak phenol oxidase activity on agar medium containing tannic acid, typical of CHV-1 infected isolates. Through sequencing of a specific region of the hypovirus genome, we found that 24 hypovirus isolates belonged to the CHV-1 subtype I and six to the CHV-1 subtype F2. The distribution of the two CHV-1 subtypes in Turkey showed a clear geographic pattern. CHV-1 subtype I was only detected in the Marmara and western Black Sea region, whereas subtype F2 was restricted to the eastern part of the Black Sea region. The effectiveness of 23 hypovirulent isolates was tested against a virulent isolate on 2–3 years old chestnut sprouts. Ten hypovirulent isolates, all infected by CHV-1 subtype I, prevented canker development by more than 80 % suggesting that they might be suitable for biological control of chestnut blight in Turkey.     

Wilt and root rot of poinsettia caused by three high-temperature-tolerant Pythium species in ebb-and-flow irrigation systems

Poinsettia plants growing in ebb-and-flow irrigation systems developed wilting and root rot during the summer growing seasons of 2010 in Gifu Prefecture and 2011 in Aichi Prefecture. Pythium species were isolated from roots with rot symptoms. The isolates were identified as P. helicoides and P. myriotylum on the basis of morphological characteristics and sequence homologies in the rDNA internal transcribed spacer regions. In pathogenicity tests, these isolates caused severe wilting and root rot. This is the first report of poinsettia root rot disease caused by P. helicoides and P. myriotylum, although P. aphanidermatum was reported as a pathogen of poinsettia root rot. To better understand these diseases, we performed an epidemiological study of three high-temperature-tolerant Pythium species, P. aphanidermatum, P. helicoides and P. myriotylum. Disease incidence as a percentage of diseased plants was greatest at 35 °C for all three species. Disease severity using the rating scale of root rot was also highest at 35 °C, particularly with high zoospore inoculum densities (100.0 zoospores/mL). Although the disease incidence and severity were reduced at lower temperatures, the three Pythium species were able to cause disease at temperatures as low as 20 °C.     

Identification of Berkeleyomyces rouxiae causing black root rot disease on cotton seedlings in New South Wales,Australia

Black root rot disease of cotton seedlings caused by Thielavioposis basicola was first reported in New South Wales (NSW), Australia in 1990. In 2018, T. basicola was reclassified into a new genus Berkeleyomyces, accommodating two closely related species: B. basicola and B. rouxiae. However, species status of cotton-T. basicola in NSW remains unsolved. Ninety-five isolates recovered from black root rot diseased cotton seedlings sampled across NSW in 2017/18 season was subjected to morphological, multigene sequencing (ITS, MCM7, RPB2), and pathogenicity assessments for their species identification. Berkeleyomyces rouxiae was accurately identified as the causal agent of black root rot of cotton.

     

Black root rot of cucurbits caused by Phomopsis sclerotioides in Japan and phylogenetic grouping of the pathogen

Although the causal agent of black root rot of Cucurbitaceae in Japan has been proposed as Phomopsis sclerotioides, the species identification of the pathogen has remained inconclusive because of a lack of spore formation. We confirmed that a Japanese isolate of Phomopsis sp. obtained from a diseased pumpkin root produced pycnidia containing α spores in sterilized bean pods. In phylogenetic analyses of rDNA-ITS regions, nine Japanese Phomopsis sp. isolates from melon, watermelon grafted onto bottle gourd, and pumpkin diagnosed with black root rot, formed a single clade with P. sclerotioides standard isolates. We identified the causal agent of the black root rot of melon, pumpkin, bottle gourd, and watermelon in Japan as P. sclerotioides and propose the Japanese name “Phomopsis-negusare-byo” for the disease. Patterns of random amplified polymorphic DNA (RAPD) of these Japanese isolates were also similar to those of P. sclerotioides, thus supporting the species identification. However, mycelial incompatibilities were found for many combinations among these P. sclerotioides isolates, suggesting some genotypic variations of this fungus in Japan at a level that the RAPD analyses cannot discriminate. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB201430 to AB201444     

Characterization of isolates of binucleate Rhizoctonia spp. associated with strawberry black root rot complex using fatty acid methyl ester (FAME) profiles

Black root rot is an important disease of strawberry caused by a complex of fungi that includes species of Rhizoctonia. In this study, a modified MIDI method (Microbial Identification System) was investigated for its utility to differentiate isolates of the three different anastomosis groups (AGs) of binucleate Rhizoctonia spp., associated with strawberry black root rot complex representing AG-A, AG-G, and AG-I. A total of 11 fatty acids were detected, and the FAME profiles for isolates of the three different AGs of Rhizoctonia spp. varied quantitatively and qualitatively. Moreover, the modified MIDI method will be a useful discriminatory tool for fungal identification and classification of the AGs of binucleate Rhizoctonia spp. associated with strawberry black root rot complex.     

Pathogenicity studies in avocado with three nectriaceous fungi,Calonectria ilicicola,Gliocladiopsis sp. and Ilyonectria liriodendri

Calonectria ilicicola, Gliocladiopsis sp. and Ilyonectria liriodendri were isolated from diseased roots of young avocado trees. Pathogenicity studies with seedlings of three avocado cultivars, Velvick, Hass and Reed, demonstrated that Calonectria ilicicola is a severe root rot pathogen, reducing the biomass of healthy roots, and reducing plant height over time. Calonectria ilicicola was re‐isolated from diseased roots. Ilyonectria liriodendri and Gliocladiopsis sp. were not pathogenic and plant height was increased after Gliocladiopsis sp. amendment compared to all other treatments in trials with cvs Velvick and Hass.     

Root and crown rot of strawberry caused by Pythium helicoides and its distribution in strawberry production areas of Japan

Pythium species were isolated from seedlings of strawberry with root and crown rot. The isolates were identified as P. helicoides on the basis of morphological characteristics and sequences of the ribosomal DNA internal transcribed spacer regions. In pathogenicity tests, the isolates caused root and crown rot similar to the original disease symptoms. Multiplex PCR was used to survey pathogen occurrence in strawberry production areas of Japan. Pythium helicoides was detected in 11 of 82 fields. The pathogen is distributed over six prefectures.     

Identification of fungal pathogens and analysis of genetic diversity of Fusarium tricinctum causing root rots of alfalfa in north-east China

Alfalfa root rot is a devastating disease complex found worldwide. Population structure and genetic diversity of fungal pathogens causing alfalfa root rot in north-east China are not well understood. In this study, 480 fungal isolates were collected from six major alfalfa-growing regions in Heilongjiang province, China. They were identified as Fusarium tricinctum, F. oxysporum, F. acuminatum, F. solani, F. equiseti, Phoma medicaginis, Plectosphaerella cucumerina, Alternaria alternata, and Chaetomium globosum and caused root rot on alfalfa in greenhouse studies. F. tricinctum was the predominant species among the isolates, and P. medicaginis and C. globosum had not previously been reported causing alfalfa root rot in north-east China. Of the 73 F. tricinctum isolates identified, the majority were moderately or highly aggressive on alfalfa. No isolate of F. tricinctum was sensitive to carbendazim (1 and 10 μg/ml), indicating that, although commonly used, it is not suitable for management of the disease in this area. F. tricinctum isolates were analysed using AFLP markers and divided into eight genetic groups with 28 pairs of primers. Analysis of molecular variance indicated significant correlation between genetic groups of F. tricinctum isolates and their geographical locations or aggressiveness. Pairwise comparison and STRUCTURE analysis also indicated that geographical locations and aggressiveness of isolates had a significant effect on population differentiation. This study provides insight into the genetic diversity and reproductive biology of F. tricinctum, enhances understanding of the population diversity of alfalfa root rot pathogens in north-east China, and facilitates development of effective strategies for managing this destructive disease complex.     

Pink root rot of squash caused by Setophoma terrestris in Japan

Pink root rot of squash (Cucurbita moschata) caused by Setophoma terrestris was found in Maebashi, Gunma Prefecture, Japan in July 2007. Cucumber grafted on the squash first developed wilt and finally blight. These symptoms followed a severe pink root rot of the squash rootstock. The fungal isolates from diseased roots were identified as S. terrestris based on morphological characteristics and nucleotide sequences. One isolate induced a similar pink root rot but not entire wilting of the cucumber vine. We propose the name “pink root rot” (koshoku-negusare-byo in Japanese) of squash for the new disease.     

<Emphasis Type="Italic">Cylindrocarpon</Emphasis> species associated with apple tree roots in South Africa and their quantification using real-time PCR

Cylindrocarpon species are known to be a component of the pathogen/pest complex that incites apple replant disease. In South Africa, no information is available on apple associated Cylindrocarpon species and their pathogenicity. Therefore, these aspects were investigated. Among the isolates recovered from apple roots in South Africa, four species (C. destructans, C. liriodendri, C. macrodidymum and C. pauciseptatum) were identified using β-tubulin gene sequencing and phylogenetic analysis. This is the first report of C. liriodendri, C. macrodidymum and C. pauciseptatum on apple trees. Cylindrocarpon macrodidymum was the most prevalent. Isolates within each of the four species were pathogenic towards apple seedlings, but varied in their virulence. With a single exception, all isolates were able to induce lesion development on seedling roots. Only 57% of the isolates, which represented all four species, were able to cause a significant reduction in seedling weight and/or height. The greatest seedling growth reductions were caused by two isolates of C. destructans, and one isolate each of C. liriodendri and C. macrodidymum. A quantitative real-time polymerase chain reaction (qPCR) method was developed for simultaneous detection of all four Cylindrocarpon species. qPCR analyses of Cylindrocarpon from the roots of inoculated seedlings showed that the amount of Cylindrocarpon DNA in roots was not correlated to seedling growth reductions (weight and height) or root rot. The qPCR method is, however, very useful for the rapid identification of apple associated Cylindrocarpon species in roots. The technique may also hold potential for being indicative of Cylindrocarpon disease potential if rhizosphere soil rather than roots are used.     

Fusarium oxysporum f. sp. lycopersici races and their genetic discrimination by molecular markers in West Mediterranean region of Turkey

Fusarium oxysporum f. sp. lycopersici (FOL) races and F. oxysporum f. sp. radicis lycopersici (FORL), the causal agents of root rot and crown rot diseases, respectively, cause serious economic losses in tomato greenhouses where production is intensive in the West Mediterranean region of Turkey. The isolates were collected from West Mediterranean region of Turkey and were characterized by specific primers based on three races (r1, r2, r3), besides pathogenicity tests in in vivo conditions Additionally, a scheme was developed using newly tested ISSR and SRAP markers to a genotyping database and to determine the possible origin of these pathogens. The present study provided new information on these pathogens based on their races and their dominant existence in this region that has not been reported before. Genetic diversity detected in the same races of the pathogen may be associated with difficulties in controlling the pathogen and a possible resistance formation effort exerted by the pathogen to chemicals used in plant protection in tomato greenhouses. Molecular analyses indicated genetic diversity in pathogen isolates identified as r3, r2 and FORL, which may be associated with abiotic stress to which the pathogens were exposed.     

Molecular characterization and pathogenicity of binucleate Rhizoctonia AG-F associated to the watermelon vine decline in Italy

In recent years, watermelon (Citrullus lanatus) has been subjected to significant losses due to vine decline in Sicily (southern Italy). During a survey conducted in 2009, the predominant fungal species associated with root rot and vine decline were Rhizoctonia spp. The most isolates were characterized as binucleate Rhizoctonia AG-F through morphological observation, nuclear condition, anastomosis tests and sequence homology of rDNA-ITS. Occasionally, R. solani was found. The pathogenicity of binucleate Rhizoctonia and virulences of different isolates were tested in growth chamber on watermelon seedlings. All isolates were pathogenic on watermelon seedlings and showed statistically significant differences on the disease incidence and severity among them. To our knowledge, this is the first report worldwide of the occurrence of pathogenic binucleate Rhizoctonia responsible for root rot and associated with watermelon vine decline.     

Fusarium commune associated with wilt and root rot disease in rice

Wilt and root rot disease in plants has been caused mainly by Fusarium species. Previous studies reported that members of the Fusarium oxysporum species complex (FOSC) were usually associated with this disease, but there has been no report of it being caused in rice by specific Fusarium species. However, in this study, Fusarium commune was identified and characterized as a causal agent of wilt and root rot disease of rice. Four Fusarium isolates (BD005R, BD014R, BD019R, and BD020R) were obtained from different parts (root, stem, and seeds) of diseased rice plants. In morphological studies, these isolates produced key characteristics of F. commune, such as long and slender monophialides, polyphialides, and abundant chlamydospores. In molecular studies, the isolates were identified as F. commune based on sequences of the translation elongation factor 1-α (TEF1) gene that had 99.7%–100% sequence identity with the reference strain F. commune NRRL 28058. The phylogenetic tree showed that all four isolates belonged to the F. commune clade. A mating type test determined that three isolates carried MAT1-2. Their teleomorph stage was still unknown. Pathogenicity assays showed that all the isolates produced wilt and root rot symptoms and the isolate BD019R was observed as the most virulent among the isolates. To our knowledge, this is the first report of F. commune causing wilt and root rot disease on rice.     

Gnomonia fragariae, a Cause of Strawberry Root Rot and Petiole Blight

Gnomonia fragariae has been occasionally listed among the fungi associated with diseased strawberry plants. However its pathogenicity has not been established. During the investigation on strawberry decline in Latvia and Sweden, a fungus was repeatedly recovered from discoloured root and crown tissues of severely stunted plants. Attempts to induce sporulation of the isolates grown on several agar media resulted in the formation of mature ascomata only on potato carrot agar and oatmeal agar. On morphological grounds and comparisons with reference herbarium specimens these isolates were identified as Gnomonia fragariae. The pathogenicity of the fungus was evaluated initially in the detached leaf assay and subsequently in three bioassays on strawberry plants. All the bioassays showed that G. fragariae was pathogenic on strawberry and capable of causing severe root rot and petiole blight. The symptoms that developed in the greenhouse experiments closely resembled those observed in the fields. The fungus did not cause rapid plant death but growth and development of inoculated strawberry plants was severely affected. To our knowledge this is the first time when pathogenicity of G. fragariae as a root rot pathogen has been clearly established. Our study shows that G. fragariae is one of the serious pathogens involved in the root rot complex of strawberry in Latvia and Sweden.     

Vegetative compatibility groups in Colletotrichum coccodes from Turkey and their aggressiveness to potato

Black dot, caused by Colletotrichum coccodes, is a common disease of potato in Turkey, affecting tuber quality and yield. The objectives of the current study were to characterize vegetative compatibility groups (VCGs) of C. coccodes isolates from three regions in Turkey, and to assess the correlation between VCGs and aggressiveness of isolates on potato. A total of 147 C. coccodes isolates were recovered from plants showing typical black dot symptoms on stolons, roots and stems. The frequency of nitrate non‐utilizing (nit) nit1/nit3 and NitM phenotypes were 79% and 21%, respectively. Complementation between nit mutants of the isolates and eight European/Israeli EU/I‐VCG tester isolates was used to characterize the VCGs. Amongst the tested isolates, 33.3% were assigned to EU/I‐VCG6, 21.8% to EU/I‐VCG8, 15.7% to EU/I‐VCG4. EU/I‐VCG1, EU/I‐VCG3, EU/I‐VCG5 and EU/I‐VCG7 were classified at 1.4%, 3.4%, 4.8% and 5.4%, frequency, respectively. No isolate was assigned to EU/I‐VCG2 group, while 21 isolates (14.3%) were not assigned to any of the EU/I‐VCGs. The pathogenicity tests indicated significant differences in aggressiveness of the isolates with respect to sclerotia density on potato tissues. The highest densities of sclerotia on roots and crown were obtained with EU/I‐VCG6 isolates and the lowest with EU/I‐VCG1, EU/I‐VCG3 and EU/I‐VCG5 isolates. The results demonstrate that there is significant VCG diversity among C. coccodes isolates from potato plants in Turkey.     

First report of the recently introduced,destructive powdery mildew <Emphasis Type="Italic">Erysiphe corylacearum</Emphasis> on hazelnut in Turkey

Hazelnut (Corylus avellana) is Turkey’s most valuable agricultural export, and an essential source of income for many families in the Black Sea Region. In spring 2013, hazelnut leaves, fruit clusters and shoots showing powdery mildew infection symptoms different from those observed previously were discovered in Giresun, Ordu and Trabzon provinces of Turkey. The disease has become epidemic throughout all hazelnut production areas spreading from east to west of the Black Sea Region over the subsequent years. Erysiphe corylacearum was identified as the causal agent of this new and highly destructive powdery mildew based on its morphological characteristics and analyses of DNA sequences of the internal transcribed spacer (ITS) and 28S regions of the ribosomal DNA. Pathogenicity of this species was examined in an infection test and proven for the first time. To our knowledge, this is the first report of E. corylacearum on Corylus avellana worldwide.     

新疆加工番茄腐霉根腐病病原鉴定及其rDNA的ITS区段分析

为了明确新疆加工番茄根腐病的病原种类,从成苗期到果实成熟期对各主栽区的病原物进行分离,并进行形态学鉴定和ITS序列分析.结果表明,从162个根腐病样中获得120个分离物,其中腐霉菌93株,占总分离物的77.5%.腐霉菌分离物鉴定为3个种,瓜果腐霉Pythium aphani-dermatum(Edson)Fitzp,56株,占腐霉分离物的60.22%;简囊腐霉P.monospermum Pringsheim,6株,占6.45%;棘腐霉P.acanthicum Drechsler,2株,占2.15%;另有29个分离物因未诱发出雄器和藏卵器而无法鉴定到种.     

Detection of black-foot disease pathogens in the grapevine nursery propagation process in Spain

Two commercial nurseries located in Comunidad Valenciana region (central-eastern Spain) were sampled in 2010 to evaluate whether the grapevine nursery propagation process could be a source of black-foot disease pathogens. Samples were taken from four sources of the propagation process: pre-grafting hydration tanks, scissors used for cutting buds, omega-cut grafting machines, and peat used for callusing. DNA from these samples was extracted and multiplex nested-PCR using primers specific for “Cylindrocarpon” pauciseptatum, Ilyonectria liriodendri and I. macrodidyma-complex (composed of I. alcacerensis, I. estremocensis, I. macrodidyma, I. novozelandica, I. torresensis, and two undescribed species) was used to identify the species present. Ilyonectria liriodendri and I. macrodidyma-complex were detected in hydration tanks, scissors, grafting machines and peat, I. macrodidyma-complex being the most frequent. Additionally, ten grafted cuttings each from five grapevine scion/rootstock combinations were collected from each nursery immediately after callusing, and again after one growing season in a nursery field. Roots of these grafted cuttings and plants were sampled to isolate the fungal pathogens. Only I. torresensis was isolated after callusing, while I. liriodendri, I. novozelandica and I. torresensis were isolated after one growing season, showing the highest incidence at this latter sampling time. Moreover, DNA was extracted from roots and analyzed as described before. Ilyonectria liriodendri and I. macrodidyma-complex were also detected at both sampling times. The use of the multiplex nested-PCR technique improved the detection of I. liriodendri and I. macrodidyma-complex from grafted cuttings and plants in both nurseries. This work shows that the grapevine nursery propagation process should be considered as a potential infection source for black-foot disease pathogens, and confirms that infections caused by Ilyonectria spp. in grapevine planting material increase markedly after one growing season in nursery fields.     

Seasonal progression of leaf rust in ‘Niagara Rosada’ grapevine in a biannual crop system in Brazil

The soil-borne fungus Fusarium oxysporum can cause both Fusarium yellows and Fusarium root rot diseases with severe yield losses in cultivated sugar beet. These two diseases cause similar foliar symptoms but different root response and have been proposed to be caused by two distinct F. oxysporum formae speciales. Fusarium yellows, caused by F. oxysporum f. sp. betae, presents vascular discoloration, whereas Fusarium root rot, due to F. oxysporum f. sp. radicis-betae, appears as black rot visible on the root surface. The aim of this work was to study the host-pathogen interaction between sugar beet lines and isolates originally characterized as Fusarium oxysporum f. sp. betae. Eight susceptible sugar beet lines, selected by the USDA-ARS (US) and UNIPD (University of Padova, Italy) breeding programs, were inoculated with three different isolates of F. oxysporum f. sp. betae, the causal agent of Fusarium yellows, representing different genetic groups. All inoculated lines developed symptoms, but severity, expressed as area under the disease progress curve (AUDPC), differed significantly (P < 0.05) among lines. Two lines from UNIPD, 6 and 9, were the most susceptible to the disease, whereas the other lines showed similar levels. The three isolates of F. oxysporum f. sp. betae differed significantly (P < 0.05) in disease severity. Five weeks after inoculation the plants were harvested and roots examined. Surprisingly, severe root rot was observed in the susceptible UNIPD lines when inoculated with all three isolates, while this symptom was never observed in the USDA germplasm. The development of this disease symptom obviously depends on the plant genotype.     

黑龙江省大豆疫霉根腐病调查与病原分离

１９９６年对黑龙江省东部和中部大豆产区２３个市、县的大豆苗期疫霉根腐病进行了调查、研究,应用ＰＢＮＩＣ疫霉选择性培养基分离大豆疫霉根腐病病原菌,从牡丹江、穆棱、林口和佳木斯豆田具疫霉根腐症状的大豆植株上分离到大豆疫霉根腐病菌,并从根腐病株上单独或与大豆疫霉菌同时分离到终极腐霉菌,研究进一步证实我国黑龙江省有大豆疫霉根腐病。调查发现,大豆疫霉根腐病和终极腐霉根腐病主要发生在土质粘重、土壤含水量高或易积水的田块。