Diplodia Seriata Research Articles

Defense Responses in Grapevine (cv. Mourvèdre) after Inoculation with the Botryosphaeria Dieback Pathogens Neofusicoccum parvum and Diplodia seriata and Their Relationship with Flowering.

As a result of the increasing economic impact of grapevine trunk diseases on viticulture worldwide, efficient and viable control strategies are urgently needed. However, understanding both plant-pathogen interactions and plant physiological changes related to these diseases is fundamental to such an achievement. In this study, we analyzed the effect of inoculation with the Botryosphaeria dieback fungal agents, Neofusicoccum parvum and Diplodia seriata, with and without inflorescence removal at the onset of G stage (separated clusters), I stage (flowering) and M stage (veraison). A measure of lesion size and real-time reverse-transcription polymerase chain reaction-based analysis were carried out. The results clearly show the importance of inflorescences in the development of lesions associated with Botryosphaeria dieback pathogens inoculated on green stems of adult vines, especially at the onset of flowering. At flowering, the biggest necroses were observed with the inflorescences present, as well as an activation of the studied defense responses. Thus, an ineffective response to the pathogen could be consistent with a possible metabolic reprogramming linked to the host phenophase.

Incidence of grapevine trunk diseases is influenced by soil, topology and vineyard age, but not by Diplodia seriata infection rate in the Tokaj Wine Region, Hungary

Grapevine Trunk Diseases (GTD) are of great importance worldwide, including Hungary, a Center European country with long wine producing history. Several GTD pathogens have been described till now in Europe, but only a few from Hungary. The presence of a GTD pathogen in the vine does not necessarily result in the immediate appearance of disease symptoms, and information on the importance of environmental factors related to disease incidence are still limited. The aim of this research was to assess the occurrence of GTD in the Tokaj Wine Region, and to determine the biotic and abiotic factors influencing disease incidence. Five vineyards within 15 km radius – each with different topology, soil types, varieties and age – were studied for three consecutive years (2013 – 2015). The incidence of GTD-infection was determined every year for each vineyard. Diplodia seriata was isolated with incidence ranging from 50 to 100%, while Diaporthe spp. were the only other – minor – GTD pathogen found. Topology and soil type appeared to be major abiotic factors affecting incidence of GTD symptom. Disease incidence was also positively correlating with the age of the vineyards, and it was in fact found to be the definitive biotic factor regarding incidence. In contrast, D. seriata infection rate appeared unrelated to disease incidence or to any of the biotic or abiotic factors investigated.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Olive Trees in Croatia

In April 2015, dieback of young olive trees (Olea europaea L., cvs. Istarska bjelica and Buza) occurred in a private orchard near Vodnjan in Istria, Croatia. From total of 50 trees, eight (16%) showed symptoms of dieback, with branches that had vascular browning in cross section and attached light brown, downwardly curled leaves. A fungus was consistently isolated from surface sterilized wood pieces of diseased branches, obtained in pure cultures on potato dextrose agar (PDA) by monohyphal isolation, and incubated at 25°C in darkness. Colonies grew slowly (1.9 mm/day), and the initial white color became gradually dark. Microscopic analysis showed hyaline, septate hypha with conidiophores bearing phialides in verticillate arrangement producing hyaline, single-celled, ovoid to ellipsoid conidia, 4.8 ± 0.7 µm × 2.4 ± 0.4 µm (n = 100). Dark parts of the colonies showed abundant dark-brown microsclerotia embedded in PDA. Solitary microsclerotia (rounded, elongated, or irregular in shape) and their aggregates were 22 to 117 µm and up to 404 µm in diameter, respectively. Based on morphology, the fungus was identified as Verticillium dahliae Kleb. (Inderbitzin et al. 2011). For molecular identification, the representative isolate MTH4 was used for PCR amplification and sequencing of its ITS region (GenBank accession no. KX061497) using the primer pair ITS5/ITS4, showing 99.8% homology to reference isolate of V. dahliae (PD322, HQ206718) (Inderbitzin et al. 2011) and 100% homology to isolate SDV1025 (KC834733) (Yu et al. 2016), confirming identification based on morphology. Pathogenicity of eight isolates obtained in this study (MTH1 to MTH8) was tested on 2-year-old olive plants (cv. Oblica), three per isolate. Their roots were gently washed with tap water to remove soil and immersed for 1 h in conidial suspension (105 spores/ml) obtained from 10-day-old colonies on PDA. Control plants were treated with sterile water only. All the plants were potted in steam-sterilized soil and kept in greenhouse at 24 ± 2°C, around 70% relative humidity, and 15 h of daylight while watered twice a week. Inoculated plants, but not controls, showed leaf chlorosis after 4 to 5 weeks, and dieback by week six. Reisolations of V. dahliae from inoculated plants were attempted as described above and were successful for all but control plants, thus satisfying Koch’s postulates. This is the first report of V. dahliae causing dieback of olive in Croatia. During this study, it was observed that approximately 15% of olive trees in neighboring orchards also showed symptoms of dieback ; therefore, V. dahliae can be suspected as more widespread in Istria than was previously known, and control measures are needed. Diplodia seriata De Not., previously found to cause similar symptoms on young olive in Croatia (Kaliterna et al. 2012), was not found in this study.

Severe Outbreak of Black Rot in Apple Fruit cv. Fuji Caused by Diplodia seriata During Pre-Harvest in Maule Region, Chile

Severe Outbreak of Black Rot in Apple Fruit cv. Fuji Caused by <i>Diplodia seriata</i> During Pre-Harvest in Maule Region, Chile

Life traits of four Botryosphaeriaceae species and molecular responses of different grapevine cultivars or hybrids

Botryosphaeriaceae is a fungal family comprising many species involved in botryosphaeria dieback, a worldwide grapevine trunk disease. Currently, the interactions between Botryosphaeriaceae species and various grapevine cultivars are poorly understood and little data is available. This study investigated various life traits of five isolates belonging to four species of Botryosphaeriaceae found in French vineyards (Diplodia mutila, Diplodia seriata, Lasiodiplodia viticola and Neofusicoccum parvum). The two species N. parvum and L. viticola exhibited the highest optimal growth temperature and the best growth rates. They were also responsible for the most extensive necrosis and cankers in three Vitis vinifera cultivars (Cabernet Sauvignon, Merlot and Ugni‐Blanc) that differed in susceptibility to botryosphaeria dieback, and in two genotypes resistant to downy and powdery mildew (RV4 and RV5). Identification of the extracellular toxins produced by isolates in culture media showed that the N. parvum isolate had a different metabolite profile from the others, producing terremutin and salicylic acid derivatives, which are known to be compounds associated with virulence. In a second step, life traits were associated with nondestructive monitoring of gene expression involved in the defence mechanisms of five grapevine cultivars and genotypes after inoculation of wood cuttings with Botryosphaeriaceae. The transcript analyses were carried out at different times and were associated with principal component analysis (PCA). Each cultivar presented a specific transcript signature and several transcripts were correlated either with the size of necrosis/cankers or with symptom reduction, thus offering useful markers for breeding or estimating the defence status of plants.

Thermal death kinetics in dry air of Diplodia seriata fungus inoculated in vitro in pruned vine shoots

The upsurge in fungal diseases that cause grapevine wood rot in young and adult plants remains a major concern for the wine sector. Since pruned vine shoots act as a reservoir of these fungi, there is a need for sanitization methods that can prevent their spread, avoiding plant reinfection. In this work, the effectiveness of dry heat treatments for the disinfection of pruning waste has been investigated. The thermal death of two inocula of Diplodia seriata fungus, associated to black dead arm disease, was studied in vitro at four different temperatures (40, 50, 60 and 65 °C) for different time periods (20, 40, 60, 90, 120 and 150 min) in a dry air incubator. Obtained data confirmed that inhibition increased as a function of temperature and exposure time. First-order and Weibull models were compared to describe D. seriata fungus survival curve kinetics, with high R 2 values (0.923 and 0.930, respectively) and high significances (p < 0.001). Both models predict that the time required to reach 99 % mortality at 65 °C would be 143 min, slightly higher than the experimentally determined minimum time of 120 min. The thermal death kinetics data reported herein has direct applications in process design and validation studies of other grapevine-pruning-waste sanitization methodologies.

Trunk Disease Fungi Associated With Diospyros kaki in South Africa.

Persimmon trees with dieback symptoms and cankers were observed in three production areas in Western Cape Province in South Africa. Isolations were made from diseased branches, cankers, and pruning wounds as well as fungal fruiting bodies on dead branches and old pruning wounds. Several trunk disease pathogens were identified based on morphological characteristics and by molecular methods, including Diaporthe eres, D. infecunda, Eutypella citricola, E. microtheca, Phaeoacremonium parasiticum, P. scolyti, P. australiense, P. minimum, Fomitiporia capensis, Fomitiporia sp., Fomitiporella sp., and Inocutis sp., which were isolated from persimmon for the first time in the world. Other first reports from persimmon in South Africa include D. foeniculina, D. ambigua, D. mutila, Diaporthe sp., Neofusicoccum australe, N. parvum, Diplodia seriata, and Eutypa lata. Pathogenicity tests conducted with all species, except the basidiomycetes, confirmed their status as possible persimmon pathogens. This is the first study to determine and identify fungi associated with diseased persimmon in South Africa. The knowledge gained in this study forms the basis for further research to determine the impact of these fungi on persimmon productivity.

Hygienization and control of Diplodia seriata fungus in vine pruning waste composting and its seasonal variability in open and closed systems

After the ban on sodium arsenite, waste management alternatives to the prevalent burning method, such as the hygienization and biodegradation in solid phase by composting, are required for the pruned material from grapevines affected by various fungi. In this work the dynamics of a fungus associated with vine decay (Diplodia seriata) during the composting process of a mixture of laying hen manure and vine pruning waste (2:1w/w) have been investigated in an open pile and a discontinuous closed biodigester. Through the optimization of the various physical–chemical parameters, hygienization of the infected waste materials was attained, yielding class-A organo-mineral fertilizers. Nevertheless, important differences in the efficiency of each system were observed: whereas in the open pile it took 10days to control D. seriata and 35 additional composting days to achieve full inactivation, in the discontinuous biodigester the fungus was entirely inactivated within the first 3–7days. Finally, the impact of seasonal variability was assessed and summer temperatures shown to have greater significance in the open pile.

Naphthalenone polyketides produced by Neofusicoccum parvum , a fungus associated with grapevine Botriosphaeria dieback

A strain of Neofusicoccum parvum isolated from declining vines was pathogenic to grapevine cultivar Inzolia in Sicily. This strain produced some metabolites in liquid medium. Crude extract, through a bio-guided purification process, yielded four naphthalenone polyketides. They were identified by comparison with spectroscopic data and optical proprieties reported in literature as: (3 S , 4 S )-7-ethyl-3,4,8-trihydroxy-6-methoxy-3,4-dihydro-1-(2 H )-naphthalenone, (3 S *, 4 S *)-3,4-dihydro-3,4,8-trihydroxy-7-(1-hydroxyethyl)-6-methoxy-1-(2 H )-naphthalenone, (4 S )-3,4-dihydro-4,8-dihydroxy-1-(2 H )-naphthalenone, named botryosphaerones D and A, isosclerone, respectively, and (3 S *,4 S *)-3,4,5-trihydroxy-1-tetralone ( 1 - 4 ). Phytotoxic activity of the isolated compounds ( 1 - 4 ) was tested on grapevine leaves at using the leaf puncture assay. All tested compounds were phytotoxic, with botryosphaerone D showing the greatest activity. The phytotoxic effects decreased when treated leaves were exposed to light. All of the metabolites did not show in vitro antifungal activity against Diplodia seriata, Lasiodiplodia mediterranea , Neofusicoccum vitifusiforme, or Phytophthora citrophthora . This is the first report of in vitro production of botryosphaerones D and A, and 3,4,5-trihydroxy-1-tetralone by N. parvum .

First Report of Black Rot on Apple Fruit Caused by Diplodia seriata in Washington State

First Report of Black Rot on Apple Fruit Caused by <i>Diplodia seriata</i> in Washington State

Study of bio-fabrication of iron nanoparticles and their fungicidal property against phytopathogens of apple orchards.

Current research trends on iron nanoparticles (FeNPs) are extensively focused because of their unique magnetic and electrical properties mostly applicable in essential medical devices. However, their fungicidal property against plant pathogens is very less known until date. Present study demonstrates a green technique for blending of FeNPs by utilising aqueous extract of neem leaf (Azadirachta indica A. Juss.) as reducing agent. Various characterisation techniques such as ultraviolet (UV)-visible spectroscopy, Fourier transform infrared spectroscopy transmission electron microscopy, scanning electron microscopy and X-ray diffraction were performed for FeNPs. The authors' results demonstrate the more cluster formation of FeNPs with size distribution of 20-80 nm. The bio-fabricated FeNPs showed enhanced biocidal activity against economically important phytopathogens of apple such as Alternaria mali, Botryosphaeria dothidea and Diplodia seriata. From the obtained results, it can be suggested that further delve into green synthesis of FeNPs can address future biotechnology concerns to limit the synthesis of FeNPs by conventional methods. Furthermore, the field study on pathogenic fungi can be an effective step to verify their agricultural applications.

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.

Incidence and etiology of postharvest fungal diseases of loquat fruit (Eriobotrya japonica (Thunb.) Lindl. cv. ‘Algerie’) in Alacant province (Spain)

‘Algerie’ is currently the most important loquat cultivar in Spain. The incidence and etiology of postharvest diseases affecting this cultivar were determined under local conditions. Latent and wound pathogens were evaluated for two consecutive seasons on commercially grown loquats from two orchards. Healthy loquats were either surface-disinfected or artificially wounded in the rind and placed in humid chambers at 20 °C for up to 5 weeks. Additionally, decay was assessed on commercially-handled loquats stored at 5 °C for up to 20 weeks. The most frequent disease was caused by Alternaria alternata, followed by Penicillium expansum. These two pathogens were present on fruit assessed for all types of infection. Moreover, decay caused by Botrytis cinerea was abundantly observed on both wounded and cold-stored fruit, while Colletotrichum gloeosporioides was frequently found on surface-disinfected fruit. Other pathogens that were observed causing latent infection to a lesser extent included Pestalotiopsis clavispora and Diplodia seriata. Common isolates were identified by macroscopic and microscopic morphology and/or DNA amplification and sequencing. Pathogenicity of selected isolates was demonstrated by fulfilling Koch’s postulates and disease development was assessed on artificially inoculated loquats stored at either 20 or 5 °C.

Botryosphariaceae species associated with stem canker, die-back and fruit rot on apple in Uruguay

Botryosphaeriaceae species are pathogens of apple and many other woody hosts worldwide. In this study, 34 apple orchards belonging to 22 farmers located in the south of Uruguay, the main apple production region, were surveyed. Stem canker, die-back and fruit rot were consistently observed, the most serious damage being due to cankers. From the 91 Botryosphaeriaceae isolates obtained, eight species belonging to the genera Botryosphaeria, Diplodia, Neofusicoccum and Lasiodiplodia were identified by means of phenotypic characteristics and phylogenetic analysis of the ITS region and the TEF-1α gene. The predominant species was Botryosphaeria dothidea (56) and the second most important was Diplodia seriata (16). Other species found were Neofusicoccum parvum (8), D. intermedia (5), N. luteum (3), D. pseudoseriata (1), N. australe (1) and Lasiodiplodia theobromae (1). D. pseudoseriata and L. theobromae were associated with apple for the first time. Most of the eight species were isolated from both stem and fruit and were distributed throughout the apple-producing region. Isolates of all species were able to cause typical symptoms in stems and fruits of apple when inoculated, but the most severe symptoms were caused by L. theobromae, N. parvum and N. luteum.

Seasonal Susceptibility of Grapevine Pruning Wounds and Cane Colonization in Catalonia, Spain Following Artificial Infection with Diplodia seriata and Phaeomoniella chlamydospora.

Diplodia seriata and Phaeomoniella chlamydospora are two fungal pathogens associated with grapevine trunk diseases worldwide. This study aimed to evaluate the period during which grapevine pruning wounds remain susceptible to fungal infection and to describe the colonization of canes artificially inoculated with these pathogens. In the first experiment, pruning wounds made in either fall or winter were separately inoculated with each pathogen at different times after pruning. Wound susceptibility to both pathogens decreased as the period between pruning and inoculation increased, from high percentages recorded in the first inoculation round (D. seriata, 97.5% and P. chlamydospora, 75%) down to approximately 10% 12 weeks after pruning. Pruning wounds remained more susceptible to D. seriata after a late pruning in winter whereas no overall seasonal changes in wound susceptibility were detected for P. chlamydospora. In the second experiment, canes were pruned by leaving two different lengths between the top node and the pruning wound before inoculations. Pathogens were recovered at different incubation periods and from different sites along the canes to estimate fungal cane colonization. A longer pruned internode made cane colonization by P. chlamydospora difficult, as indicated by fungal recoveries lower than 10% at the lowest recovery site, whereas D. seriata was less inhibited.

Botryosphaeriaceae species associated with wood diseases of stone and pome fruits trees: symptoms and virulence across different hosts in Uruguay

Apple, pear and peach orchards are usually planted in close proximity, and they might share pathogenic fungal species affecting different plant organs. In particular, species of Botryosphaeriaceae have been indicated as responsible for wood diseases in these fruit species. Symptoms frequently observed in Uruguay include papyraceous cankers in apple trunks, dead shoots in pears, and gummosis in peach shoots and branches. Symptomatic tissues of these species were sampled, and fungi resembling species within Botryosphaeriaceae were isolated and identified based on phylogenetic analysis of the internal transcribed spacer region (ITS), part of the translation elongation factor 1-α gene (EF1-α) and part of the RNA polymerase II subunit gen (RPB2). Pathogenicity of selected isolates was assessed under laboratory and field conditions. Botryosphaeria dothidea, Diplodia seriata and Neofusicoccum parvum were isolated from the three hosts from symptomatic tissues. However, only N. parvum proved to be pathogenic when inoculated on trees of all three species, while D. seriata showed pathogenicity towards apple and peach trees only. Diplodia mutila and N. australe were isolated less frequently and not from all the host species. However, they were the most virulent based on lesion length on all tested plant species. The fact that fungal isolates obtained from one host could cause disease symptoms also in some of the alternative hosts suggests that cross infections are possible between apple, pear and peach trees. Finally, Diplodia pseudoseriata in Prunus persica and Diplodia mutila in Pyrus communis, are new records for these hosts in Uruguay.

Reproducing Botryosphaeria Dieback Foliar Symptoms in a Simple Model System.

Botryosphaeria dieback is a grapevine trunk disease with a worldwide distribution associated with Diplodia seriata and Neofusicoccum parvum, among several other Botryosphaeriaceae species. The aforementioned xylem-inhabiting fungi cause wood lesions and leaf and berry symptoms, and eventually lead to the death of the plant. The aim of this work was to develop a simple model system to reproduce the foliar symptoms caused by D. seriata and N. parvum to better characterize fungal pathogenicity and determine the mechanisms involved in symptom development. Green stems of grafted 'Aragonez' grapevine cuttings were inoculated with three isolates of N. parvum and two isolates of D. seriata with different degrees of virulence and the experiment was repeated four times from 2011 to 2014. Three months after inoculation, the lesions associated with N. parvum were larger than those associated with D. seriata. Similarly, 8 months after inoculation, the percentage of plants showing foliar symptoms was greater in the N. parvum treatments than in the D. seriata treatments. During the emergence of foliar symptoms, plant stress-related responses were modulated in green stems and leaves, especially a downregulation of superoxide dismutase (SOD) and fasciclin-like arabinogalactan protein (fascAGP) and an upregulation of stilbene synthase (STS) genes with an accumulation of phenolics. In conclusion, the simple model system developed allowed a good characterization of isolate pathogenicity and correlation with foliar symptoms of Botryosphaeria dieback, namely spots on leaf margin and blade.

Black Wilt of Hop (Humulus lupulus) Caused by Diplodia seriata in New York State

Black Wilt of Hop (<i>Humulus lupulus</i>) Caused by <i>Diplodia seriata</i> in New York State

Phylogeny, morphology and pathogenicity of Botryosphaeriaceae, Diatrypaceae and Gnomoniaceae associated with branch diseases of hazelnut in Sardinia (Italy)

Severe trunk and branch diseases of hazelnut trees have recently been observed in several groves in Sardinia (Italy). Since there is little information about the aetiology of these diseases and given the high ecological and economic importance of these agro-systems, an in-depth study was carried out. From autumn 2012 to spring 2014, sixty samples of twigs and branches of hazelnut trees showing exudates and different types of canker (sunken with wedge-shaped necrotic sector, open canker and Cytospora canker) were collected in the main hazelnut growing area in the centre of the island. Based on morphology, colony appearance and DNA sequence data, seven species belonging to four genera and three families were isolated and identified. These included Diplodia sapinea, D. seriata, Dothiorella iberica, Do. parva and Do. symphoricarposicola (Botryosphaeriaceae), Gnomoniopsis smithogilvyi (Gnomoniaceae) and Anthostoma decipiens (Diatrypaceae). In addition, two new species namely Diaporthella cryptica sp. nov. and Dothiorella omnivora sp. nov. are described. Pathogenicity trials carried out on wounded hazelnut branches showed that three species, Anthostoma decipiens, Diaporthella cryptica and Diplodia seriata are aggressive pathogens on hazelnut. Results obtained have allowed us to clarify, almost a century after its first description, the aetiology of the disease known as Cytospora canker of hazelnut and to reveal the existence of three evolutionarily distinct lineages for its causal agent A. decipiens. The diversity of fungal pathogens associated with twig and branch cankers of hazelnut is greater than previously recognised and further studies are necessary to determine the exact role played by each species and their possible synergistic interaction.

Identification and characterization of fungi associated with internal wood lesions and decline disease of willow and poplar trees in Iran

SummaryDuring the study of fungal trunk pathogens associated with urban trees decline in Shiraz (Iran), a serious decline of willow and poplar trees was observed. Therefore, an investigation was conducted on these trees in some areas of this city during spring and summer 2012 and 2013, to determine the main fungal trunk pathogens associated with these ornamental plants. Plant materials were collected from trees exhibiting disease symptoms such as yellowing, shoot canker, shoot dieback, defoliation and internal wood necrosis and decayed wood. Fungal isolations were made from discoloured or decayed wood tissue onto 2% malt extract agar (MEA) amended with streptomycin sulphate. Nine species,Fomes fomentarius,Diplodia seriata,Lasiodiplodia theobromae,Dothiorella sarmentorum,Neoscytalidium hyalinum,Diatrype whitmanensis,Phaeoacremonium rubrigenum,P. aleophilumandP. parasiticum, were identified based on morphology and DNA sequence comparisons. Pathogenicity tests were performed on detached shoots of willow and poplar trees under greenhouse conditions.Lasiodiplodia theobromaecaused the longest lesions on willow. On poplar shoots, the longest lesions were caused byP. parasiticum.Diplodia seriataproduced the smallest lesions on both woody hosts. First reports from willow wood includeP. parasiticum, P. rubrigenum, D. whitmanensis, L. theobromae, D. seriataandN. hyalinum,while new reports from poplar wood includeP. parasiticumandDo. sarmentorum. Based on our knowledge, this is also the first report ofD. whitmanensisin Iran.