Dieback Symptoms Research Articles

Development of a Multiplex PCR Assay to Detect Neofusicoccum parvum and Botryosphaeria dothidea in Walnut.

In walnut orchards, frequent symptoms of cankers and dieback (fruit blight, twig and branch cankers up to tree death) are caused by different agents, in particular by Botryosphaeriaceae, primarily Neofusicoccum parvum and Botryosphaeria dothidea. This study aimed at developing a sensitive, rapid, specific and internally controlled multiplex PCR assay for the detection of these species. The ability of the multiplex PCR, with an internal inhibition control (i.e. E. coli DNA), to specifically and successfully detect members of the two targeted species was validated using 11 different isolates for each fungal target (inclusivity) and 20 tree-associated fungal species different from B. dothidea or N. parvum (exclusivity). Applicability to plant materials was further investigated and showed an absence of amplification for asymptomatic husk or twig samples while the amplification profiles of symptomatic tissues ranged from no amplification to amplification of both species, in correlation with the observed fungal contamination level. In conclusion, we developed a rapid diagnostic tool for simultaneous detection of two major fungal pathogens of walnut. Although this protocol was tailored for walnut husks and twigs, applicability to any plant sample contaminated by these pathogens can be considered.

First report of Botryosphaeria dothidea causing root rot of sugar beet in Serbia.

Botryosphaeria dothidea (Moug.:Fr.) Ces. & De Not. is predominantly recognized as a pathogen of various woody plants, inducing symptoms of stem canker, dieback, and fruit rot worldwide. However, sporadic reports suggest its impact on field crops, including B. dothidea associated with stem canker in soybean and tobacco (Bian et al. 2015; Chen et al. 2021), as well as B. quercuum on sugar beet (Alfieri et al. 1984). In September 2023, during a survey of root rot pathogens of sugar beet (Beta vulgaris L.) in Rimski Šančevi, Serbia (N 45°19´57″; E 19°49'58″), 3% of collected samples exhibited root rot symptoms. Externally, the lesions exhibited a dark brown coloration. On cross-section, the tissue displayed a gradient of discoloration ranging from light to dark brown throughout the roots. The roots were completely rotted. From these samples, two fungal isolates (SR28/II and SR4/III) were obtained from rotted internal root fragments, after washing, surface disinfection (70% ethanol), and plating on potato dextrose agar (PDA). Colonies on PDA were fluffy with abundant aerial mycelium, surface light to dark grey-brown, reverse black in the centre and grey-brown towards the irregular margin, after 7 days at 25°C in the dark. To induce sporulation, isolates were cultivated on 2% water agar with pine needles and incubated under continuous near ultraviolet (NUV) light at room temperature for 30 days. Conidia were hyaline, aseptate, fusiform, subtruncate at the base, subobtuse at the apex, and measured (19.56-) 24.12 - 26.31 (-28.99) x (5.13-) 5.94 - 6.54 (-7.44) µm (mean 25.06 x 6.26 µm, n=100), consistent with description of B. dothidea (Phillips et al. 2013). For molecular identification, DNA was extracted from mycelium of 7-day-old cultures, and internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha gene (TEF) and partial β-tubulin gene (TUB) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Multiple sequence alignment and BLAST analyses showed that our isolates had 100% sequence similarity with reference isolates of B. dothidea, including ex-type isolate CBS 115476 (= CMW 8000) in ITS (AY236949), TEF (AY236898), and TUB (AY236927). Maximum likelihood phylogeny of concatenated sequences confirmed the identity of the isolates as B. dothidea. Sequences of ITS, TEF and TUB of isolate SR4/III were submitted to GenBank under accession numbers PP908658, PP911334, PP911333, respectively. The pathogenicity of obtained isolates was assessed on 3-month-old sugar beet plants grown in sterile substrate in the greenhouse. For inoculation, the upper parts of the roots were wounded (10x3 mm) 1 cm above the substrate, using a sterilized nail, and 2x2 mm mycelial plugs of 7-day-old culture grown on PDA were inserted and sealed with parafilm. Control plants were inoculated with sterile PDA plugs. Six plants were used per isolate and for control. After 3 weeks of incubation, the inoculated plants were removed from the substrate and brown rot symptoms were observed upon cross-sectional examination, whereas control roots remained asymptomatic. The pathogen was successfully reisolated and morphologically identified as B. dothidea, fulfilling Koch's postulates. The production of sugar beet, an important industrial crop in Serbia valued for its sugar content, is imperiled by various pathogens. To our knowledge, this is the first report of B. dothidea causing root rot of sugar beet in Serbia and the world. Given its widespread distribution on other hosts and potential aggressiveness, the presence of B. dothidea in sugar beet should not be neglected.

Characterization of Neopestalotiopsis species associated with lingonberry (Vaccinium vitis-idaea L.) diseases in Canada

Lingonberry (Vaccinium vitis-idaea L.) is a dwarf shrub that is native to Northern climates. The berries are harvested from wild plants and the disease incidence in the crop is generally low. However, disease pressure may increase since the surface area dedicated to the crop is expected to expand in the future. In 2021 and 2022, brown necrotic leaves and stem dieback symptoms were observed on lingonberry plants grown at the St. John’s Research and Development Center, Newfoundland and Labrador, Canada. Pathogens were isolated from the diseased plant tissues and identified as Neopestalotiopsis rosae (n = 2) and Neopestalotiopsis zimbabwana (n = 2) using colony and conidia morphology as well as sequencing of the Internal Transcribed Spacer (ITS), beta-tubulin gene (ß-tub), and the translation elongation factor 1-alpha gene (tef1). Pathogenicity assays in detached lingonberry and strawberry leaves as well as lingonberry plants indicated that all four isolates were able to cause disease. This is the first report of N. rosae and N. zimbabwana causing leaf spot and stem dieback of lingonberry. Further monitoring of this disease is important to understand its prevalence in berry crops, as Neopestalotiopsis spp. in Canada have been reported to occur since 2018 in strawberry and other small fruits. Control measures and strategies are required to mitigate the potential risk from the disease, as there are no registered fungicides available to the growers.

Impacts of phosphite treatment on Phytophthora community assemblages and inoculum abundances in Phytophthora-infected forest soil

Phytophthora are causing declines in forest tree species worldwide and the chemical control treatment, phosphite, is the only treatment consistently shown to provide some protection to natural ecosystems from Phytophthora diseases. Phosphite inhibits Phytophthora growth and sporulation whilst boosting defence responses in the plant host. It is unclear, however, the extent of the impact of phosphite on Phytophthora species assemblages and inoculum abundances in soil around trees following treatment within natural ecosystems. In New Zealand, kauri (Agathis australis), an endemic and threatened foundation species, suffers from a dieback disease primarily caused by Phytophthora agathidicida. Phosphite is applied by trunk injection to kauri and has been shown to improve resin ‘bleed’ symptoms from basal trunk lesions and to promote recovery of thinned canopies. Phytophthora community and inoculum abundance were investigated in response to phosphite treatments at two field sites (Huia and Waitoki) in infected kauri stands in Auckland, New Zealand. At Huia, soil sampling and tree health surveying were conducted in November 2023 on trees treated with phosphite in 2012 as part of an earlier study. At Waitoki, the response to phosphite treatment was monitored 6 and 18 months following treatment. Phytophthora species were detected using soil baiting and metabarcoding of environmental DNA (eDNA) from soil and quantified with qPCR of root and soil DNA. Three species were detected with soil baiting (P. agathidicida, P. cinnamomi, and P. multivora) and two additional species with metabarcoding (P. pseudocryptogea, and an unknown clade 7 species similar to P. europaea). Phytophthora cinnamomi was the most abundant species, followed by P. agathidicida. Both species were more likely to occur together than by chance alone and were associated with declining tree health. The P. europaea-like species was in approximately 50% of the samples and was less likely to occur in roots with poorer health, or in association with P. agathidicida. The abundance of P. agathidicida inoculum was lower in the soil around the phosphite-treated trees than around the untreated control trees 1.5 years after treatment at Waitoki. Phosphite halted the lateral expansion of basal resin bleeds, and resin viscosity was reduced. Not only did phosphite treatments improve kauri dieback symptoms, but the phosphite treatments potentially had a direct impact on the epidemiology of the disease by reducing inoculum load around treated trees, with direct implications for disease management as an effective way to protect uninfected areas and minimise the spread of inoculum from infested zones.

Identification of fungal species associated with gall oak (Quercus infectoria Oliv.) decline in Iran.

The gall oak (Quercus infectoria Oliv.) tree is one of the most important and valuable forestry species in the Northern Zagros forests in the west of Iran. Gall oak decline is considered to be one of the most important diseases currently affecting the oak Zagros forests in Iran. The main objective of the present study, conducted in the years 2021-2023, was to investigate the possible role of fungi as causative agents of gall oak dieback in the Zagros forests of Iran. Wood samples were taken from gall oak trees showing canker, dieback, and internal wood discoloration symptoms. Fungal isolates recovered from gall oak trees were identified based on cultural and morphological characteristics, as well as phylogenetic analyses using DNA sequencing of the internal transcribed spacer region of rDNA (ITS) and partial beta-tubulin (tub2). Achaetomium aegilopis, Alternaria tenuissima, Apiospora intestine, Botrytis cinerea, Coniochaeta sp., Coniothyrium palmarum, Coniothyrium sp., Cytospora rhodophila, Dialonectria episphaeria, Diatrype sp., Diatrypella macrospora, Endoconidioma populi, Fonsecazyma sp., Fusarium ipomoeae, Jattaea discrete, Kalmusia variispora, Microsphaeropsis olivacea, Neoscytalidium dimidiatum, Paecilomyces lecytidis, Paramicrosphaeropsis eriobotryae, Paramicrosphaeropsis ellipsoidea, and Seimatosporium pezizoides were identified from diseased trees. Pathogenicity tests were performed by artificial inoculation of excised branches of healthy gall oak trees under controlled conditions and evaluated after 35 days by measuring the discolored lesion length at the inoculation site. N. dimidiatum was the most virulent species and caused the longest wood necrosis within 35 days of inoculation. In the greenhouse test, only some species induced typical symptoms of canker. All isolated fungi are reported for the first time on gall oak trees in the world.

Association of water stress and Fusarium solani exacerbated Dalbergia sissoo dieback disease.

Globally, there is a growing concern about tree mortality due to harsh climates and changes in pest and disease patterns. However, experimental studies on the interactions between biotic and abiotic stresses in plants are relatively scarce. In this study, we investigated the interaction between Fusarium solani and water-stressed Dalbergia sissoo saplings. We postulated that under drought conditions, sissoo plants would become more susceptible to dieback infestation. Five fungi, including Fusarium oxysporum, Curvularia lunata, Cladophialophora carrionii, Alternaria alternaria, and Fusarium solani, were isolated from an old shisham tree showing advanced symptoms of dieback infestation. These fungi were identified based on their ITS sequence homology and spore characteristics. Dieback development was more pronounced in plants experiencing water stress, regardless of their predisposition or whether it occurred simultaneously. Lesions were more noticeable and longer in predisposed saplings (3.8cm), followed by simultaneous (2.4cm) and much smaller lesions in seedlings that were inoculated and well-watered (0.24cm). Progressive browning of the upper leaves, which lowers sapling height in predisposed, simultaneous, and well-watered inoculated saplings to 8.09 inches, 5.93 inches, and 17.42 inches, are typical dieback symptoms. Water stress causes the loss of chlorophyll a, b, and carotenoids, which reduces stomatal conductance, transpiration rate, and photosynthetic activity, leading to poor development and mortality. Similarly, predisposed, simultaneous, and well-watered inoculated seedlings expressed increased activity of CAT (22.57, 18.148, and 9.714 U/mg) and POD (3.0, 4.848, 1.246 U/mg), to reduce the damage caused by elevated levels of H2O2 expression. It is concluded that water stress is the main cause of dieback in shisham saplings that subsequently disposed of infected seedlings to secondary agents such as fungi and insects in the advanced stages of the dieback with prolonged drought stress. The lack of dieback in native populations is attributed to the absence of several ecological stresses, including water stress, extended droughts, waterlogging, and salinity. This study emphasizes the need for additional research into the effects of abiotic factors linked with fungal diseases on the long-term production and management of D. sissoo in Pakistan.

Botryosphaeriaceae species associated with branch dieback and decline of macadamia trees in South Africa

Botryosphaeriaceae species are important latent pathogens causing diseases on trees utilized in forestry and agriculture. In recent years, there has been an increase in the incidence and severity of branch dieback and decline on macadamia trees in South Africa, and species of Botryosphaeriaceae have been considered as a possible cause. Although botryosphaeria dieback has been well-studied in Australia, there is little information regarding these fungi on Macadamia in South Africa. The aims of this study were consequently to (i) identify species of Botryosphaeriaceae from Macadamia branches from main production regions in South Africa, (ii) compare the diversity of species between symptomatic and asymptomatic branches, as well as between different growing regions, (iii) and to consider their relative importance in causing dieback. Eight species and three putative hybrids of the Botryosphaeriaceae were identified based on a phylogenetic comparison of sequence data from the ITS rDNA, tub2, tef-1α and rpb2 loci. These included an unidentified Diplodia sp., and Lasiodiplodia sp., as well as L. gilanensis, L. theobromae, L. pseudotheobromae, Neofusicoccum kwambonambiense, N. luteum, N. parvum and three hybrid species. The unidentified species of Diplodia., Lasiodiplodia sp., L. gilanensis, and N. kwambonambiense are reported for the first time on Macadamia in South Africa. All species showed a potential to cause branch dieback symptoms, with species of Neofusicoccum identified as the most aggressive species. This study revealed a high level of diversity of Botryosphaeriaceae species and illustrates their potential as causal agents of dieback on Macadamia in South Africa.

Diplodia seriata Isolated from Declining Olive Trees in Salento (Apulia, Italy): Pathogenicity Trials Give a Glimpse That It Is More Virulent to Drought-Stressed Olive Trees and in a Warmth-Conditioned Environment.

The fungi Botryosphaeriaceae are involved in olive declines in both the world hemispheres and in all continents where this species is cultivated. In Salento (Apulia, Italy), the Botryosphaeriaceae Neofusicoccum mediterraneum and N. stellenboschiana have been reported as the agents of a branch and twig dieback that overlaps with olive quick decline syndrome caused by Xylella fastidiosa subsp. pauca. In this study, we report the finding of Diplodia seriata, another Botryosphaeriaceae species, in Salento in Xylella fastidiosa-infected olive trees affected by symptoms of branch and twig dieback. Given that its presence was also reported in olive in the Americas and in Europe (Croatia) with different degrees of virulence, we were prompted to assess its role in the Apulian decline. We identified representative isolates based on morphological features and a multilocus phylogeny. In vitro tests showed that the optimum growth temperature of the isolates is around 25-30 °C, and that they are highly thermo-tolerant. In pathogenicity trials conducted over eleven months, D. seriata expressed a very low virulence. Nonetheless, when we imposed severe water stress before the inoculation, D. seriata significatively necrotized bark and wood in a time frame of 35 days. Moreover, the symptoms which resulted were much more severe in the trial performed in summer compared with that in autumn. In osmolyte-supplemented media with a water potential from -1 to -3 Mpa, the isolates increased or maintained their growth rate compared with non-supplemented media, and they also grew, albeit to a lesser extent, on media with a water potential as low as -7 Mpa. This suggests that olives with a low water potential, namely those subjected to drought, may offer a suitable environment for the fungus' development. The analysis of the meteorological parameters, temperatures and rainfall, in Salento in the timeframe 1989-2023, showed that this area is subjected to a progressive increase of temperature and drought during the summer. Thus, overall, D. seriata has to be considered a contributor to the manifestation of branch and twig dieback of olive in Salento. Coherently with the spiral decline concept of trees, our results suggest that heat and drought act as predisposing/inciting factors facilitating D. seriata as a contributor. The fact that several adverse factors, biotic and abiotic, are simultaneously burdening olive trees in Salento offers a cue to discuss the possible complex nature of the olive decline in Salento.

Identification and Characterization Colletotrichum spp. Causing Mango Dieback in Indonesia.

Dieback disease in mango trees has been observed in Indonesia, particularly in Java Island, with the causal agent remaining unidentified. One of the important pathogens that are responsible for causing mango dieback is Colletotrichum. Field surveys were conducted in various mango cultivating areas in Java Island, Indonesia to assess prevalence of Colletotrichum as dieback disease pathogen. Eleven Colletotrichum isolates were recovered from symptomatic dieback twigs and morphologically characterized. Genetic diversity fingerprint analysis was carried out using rep-PCR. Phylogenetic analysis identified isolates as belonging to Colletotrichum asianum and Colletotrichum cairnsense using partial sequences of four gene regions, including ITS, ACT, GAPDH, and TUB2. Pathogenicity tests on mango seedlings cv. Arumanis showed that all fungal isolates were responsible for causing dieback symptoms. Subsequently, symptomatic tissue was reisolated to fulfill Koch's Postulate. This study represented new funding for two species of Colletotrichum causing mango dieback in Indonesia.

Dieback of royal poinciana (Delonix regia) trees induced by Alternaria tenuissima and its impact on photochemical efficiency of photosystem II

In Saudi Arabia, poinciana (Delonix regia) trees are afflicted by dieback disease, manifesting symptoms that can lead to complete defoliation and, ultimately, the demise of trees in advanced stages. To investigate the dieback symptoms, samples were collected from branches at the transition zone. Identification of fungal isolates involved the assessment of colony and spore morphology, as well as PCR amplification and sequencing of the internal transcribed spacer (ITS) gene regions. The predominant fungus isolated was Alternaria tenuissima. Chlorophyll a fluorescence quenching and polyphasic fluorescence induction kinetics were conducted to assess the photochemical and nonphotochemical processes of Photosystem II (PSII) in stem barks affected by dieback. The dieback disease significantly reduced chlorophyll content, potentially hindering PSII performance in terms of maximum photochemical efficiency, quantum yield, electron flux, and photoprotection. These findings suggest that compromised photochemistry and photoprotection render poinciana trees inoculated by dieback less efficient in utilizing light energy and more susceptible to photoinhibition. Additionally, this study implies the use of non-invasive chlorophyll fluorescence techniques as a phenotyping tool for assessing photoprotective and photoinhibitory responses of stems to dieback stress.

First Report of Phytopythium litorale Causing Root Rot on Kousa Dogwood in Tennessee and the United States.

Kousa dogwood (Cornus kousa) is an economically important woody ornamental crop that exhibits creamy, white, pointed bracts in late spring, and reddish to pink drupe fruits in late summer and fall. It bears shiny dark green leaves that become reddish-purple to scarlet in the fall. In August of 2023, 3-year-old container grown C. kousa var. chinensis plants in a commercial nursery in Warren Co., Tennessee, exhibited severe yellowing, dieback and root rot symptoms (Fig. 1a and 1b). Dark brown to black lesions were observed in the root and crown region of the plants. Disease severity was 40% to 60% of root area affected, and disease incidence was approximately 40% of 1,000 plants. Surface-sterilized (10% NaOCl: 1 min) symptomatic root tissues were plated on V8-PARPH and incubated at 25°C. Sparse aerial mycelium, showing a distinct rosette or faint radiate to chrysanthemum colony pattern, was observed within four days of incubation (Fig. 2). All isolates produced ovoid or subglose, papillate, and proliferating sporangia in grass blade water cultures (Derviş et al. 2020). Sporangia measured as 19.18 to 24.80 µm X 18.08 to 22.16 µm (n = 50) with a length/width ratio of 1.06 to 1.11. Zoospores observed were between 7.07 to 9.98 µm in diameter (n = 50). Oogonia and oospores were not produced. The ribosomal internal transcribed spacer (ITS) and large subunit (LSU), as well as mitochondrial cytochrome oxidase subunit II (COX-II) genetic markers were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), NL1/NL4 (Baten et al. 2014), and cox2-F/cox2-RC4 (Choi et al. 2015), respectively. The ITS, LSU, and COX-II sequences of isolates FBG6343, FBG6344 (ITS: PP458373 and PP461387; LSU: PP461390 and PP461391; COXII: PP477112 and PP477113) were 100% identical to those of MN306118, HQ643386, and MN206732, respectively. Based on the morphology (Nechwatal and Mendgen 2006) and sequence data, the isolates were identified as Phytopythium litorale (Nechw.) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque. The pathogenicity test was performed on 3-year-old C. kousa var. chinensis plants grown in a 3-gal container to fulfill Koch's postulates. Kousa dogwood plants were drench inoculated (800 ml/plant) with a pathogen slurry (two plates of 7-day-old culture/liter) of isolates FBG6343 and FBG6364 (five plants per isolate). Control plants were drenched with agar slurry without the pathogen. The study was conducted in a greenhouse maintained at 21 to 23°C and 70% relative humidity with a 16-h photoperiod and irrigated twice a day for 2 min using an overhead irrigation system. Forty-five days after inoculation, plants showed dieback symptoms, and dark brown lesions developed in the roots of all inoculated plants. Isolates with morphology and sequences identical to those of FBG6343 and FBG6364 were recovered from root tissues of all inoculated plants. All control plants remained symptom-free, and P. litorale was not isolated from the root tissue. Previously, P. litorale was reported to cause disease on apple, kiwi, planatus, and rhododendron (Derviş et al. 2020; Li et al. 2021; Mert et al. 2020; Polat et al. 2023). To our knowledge, this is the first report of P. litorale causing root rot of kousa dogwood in Tennessee and the United States. Identification of this pathogen as the causal agent is crucial to developing timely management practices.

BEECH FORESTS OF THE "VYZHNYTSKYI NATIONAL NATURAL PARK": CURRENT SANITARY CONDITION AND PROBLEMS OF PRESERVATION

The beech primeval forests of the Vyzhnytskyi National Natural Park are a unique cultural heritage and play a special role for the Carpathian region and Ukraine as a whole, therefore they are strictly protected in the territories of the nature reserve fund, performing ecological, educational and scientific-cognitive functions. In this regard, monitoring the current state of the beech forests of the Vyzhnytskyi National Natural Park and highlighting the problem of their preservation becomes relevant. In the course of conducting research, classical and modern methods of forestry research were used, as well as specific methods of phytosanitary monitoring and comparative ecology. The massif of primeval beech forests of the Vyzhnytskyi National Natural Park is characterized by a remarkable diversity of fungi. We identified fruiting bodies of wood-destroying fungi on the trunks of beech trees: Fomes fomentarius (L. ex Fr.) Gill., Daedalea quercina L. ex Fr., Phellinus igniarius (L.: Fr.) Quel, Laetiporus sulphureus (Bull.: Fr.) Bond. et Sing., Coriolus versicolor (L.) Quél., Stereum hirsutum (Willd.) Pers., Daldinia concentrica (Bolton) Ces. & De Not., Аrmillariella mellea (Vahl) P.Karst., and Pleurotus ostreatus (Jacq.) P.Kumm. Groups of Coprinus micaceus (Fr.) and Hypholoma capnoides (Fr. ex Fr.) Kumm were noted near rotten beech stumps. We also came across a unique find the invasive red-listed Clathrus archeri (Berk.) Dring. In the course of surveys, fruiting bodies of typical representatives of the marsupial class Xylaria polymorpha (Pers.) Grev., and Xylaria hypoxylon (L.: Fr.) Grev). Representatives of xylophilic ascomycetes were identified on the dead trunk of a European beech Bisporella citrina (Batsch) Korf et S.E. Carp. Symptoms of dieback of central shoots and side branches, dieback of European beech bark, which is directly related to the vital activity of Nectria ditissima Tul., were recorded. & C.Tul. Cumulative manifestations of infectious and non-infectious pathologies were noted. The problems of preserving the beech forests of the Vyzhnytskyi National Natural Park should include: intensive and uncontrolled economic activity; irresponsible behavior of tourists (arson, mechanical damage, littering, poaching, etc.); construction of highways and tourist centers in the immediate vicinity of primeval forests; damage to forest stands due to atmospheric air pollution, climate change; significant spread of pests or pathogens of forest diseases; danger of forest fires; recreational load and livestock grazing. Given the role and importance of the beech forests of the Vyzhnytskyi National Natural Park, a comprehensive study of the forest systems is necessary to preserve these unique resources.

Lasiodiplodia theobromae and Lasiodiplodia brasiliense causing dieback and rot fruit of jackfruit tree in Brazil

The jackfruit tree (Artocarpus heterophyllus) is a tropical species of significant importance, widely distributed in various geographical regions of Brazil, where its fruits, known as jackfruit, are consumed fresh. Between 2019 and 2023, in three distinct geographical regions of Brazil, a 100% incidence of black rot was observed in jackfruit fruits. Additionally, symptoms of dieback were identified in jackfruit trees in these regions. Faced with this issue, the present study aims to identify the etiological agents associated with these two symptoms, employing morphological characterization, nucleotide sequencing, and inoculations in fruits and seedlings. Through sequencing of the ITS, TEF, and TUB2 regions, the isolates were grouped with references of Lasiodiplodia theobromae and L. brasiliense. The inoculated fungi were reisolated, maintaining the same morphological characteristics. This study constitutes the first report of L. theobromae and L. brasiliense as causative agents of fruit rot and dieback in jackfruit in Brazil.

Determining the Main Infection Courts in Sweet Cherry Trees of the Canker Pathogens Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata.

The major fungal canker pathogens causing branch dieback of sweet cherry trees in California include Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata. These pathogens have long been known to infect cherry trees mainly through pruning wounds. However, recent field observations revealed numerous shoots and fruiting spurs exhibiting dieback symptoms with no apparent pruning wounds or mechanical injuries. Accordingly, this study was conducted to assess the incidence of the three pathogens in symptomatic terminal shoots and dying fruiting spurs, in addition to the wood below pruning wounds in branches. Surveys were conducted in five sweet cherry orchards across three counties in California. We also investigated the possibility that leaf scars, bud scars, and wounds resulting from fruit picking could serve as infection courts for Cal. pulchella, Cyt. sorbicola, and E. lata by means of artificial inoculations in the field. Orchard surveys revealed that Cal. pulchella had the highest pathogen incidence below pruning wounds in branch samples, followed by Cyt. sorbicola and E. lata. Among terminal shoots with dieback symptoms and dying fruiting spurs, Cyt. sorbicola was the most prevalent, followed by Cal. pulchella. Results from field inoculations indicated that fruit-picking wounds could serve as important infection courts for Cal. pulchella, Cyt. sorbicola, and E. lata, with average pathogen recovery of 41.5, 63, and 36.2%, respectively. Results also indicated that leaf and bud scars could serve as an entry site for Cyt. sorbicola, although recovery was relatively low. The present study is the first to identify harvest-induced wounds on fruiting spurs of sweet cherry as an important infection court of Cal. pulchella, Cyt. sorbicola, and E. lata.

Fungal Trunk Diseases Causing Decline of Apricot and Plum Trees in the Czech Republic.

Fungal trunk diseases (FTDs) have been a significant threat to the global stone fruit industry. FTDs are caused by a consortium of wood-decaying fungi. These fungi colonize woody tissues, causing cankers, dieback, and other decline-related symptoms in host plants. In this study, a detailed screening of the fungal microbiota associated with the decline of stone fruit trees in the Czech Republic was performed. The wood fragments of plum and apricot trees showing symptoms of FTDs were subjected to fungal isolation. The partial internal transcribed spacer region, partial beta-tubulin, and translation elongation factor 1-α genes were amplified from genomic DNA extracted from fungal cultures. All isolates were classified, and the taxonomic placement of pathogenic strains was illustrated in phylogenetic trees. The most abundant pathogenic genus was Dactylonectria (31%), followed by Biscogniauxia (13%), Thelonectria (10%), Eutypa (9%), Dothiorella (7%), Diplodia (6%), and Diaporthe (6%). The most frequent endophytic genus was Aposphaeria (17%). The pathogenicity of six fungal species (Cadophora daguensis, Collophorina africana, Cytospora sorbicola, Dothiorella sarmentorum, Eutypa lata, and E. petrakii var. petrakii) to four Prunus spp. was evaluated, and Koch's postulates were fulfilled. All tested isolates caused lesions on at least one Prunus sp. The most aggressive species was E. lata, which caused the largest lesions on all four tested Prunus spp., followed by E. petrakii var. petrakii and D. sarmentorum. Japanese plum (Prunus salicina) and almond (P. amygdalus) were the most susceptible hosts, while apricot (P. armeniaca) was the least susceptible host in the pathogenicity trial.

Characterization and pathogenicity of Pleurostoma richardsiae causing decline of mango trees in Southern Italy

Mango trees (Mangifera indica) showing symptoms of twig and branch dieback, internal wood necroses, and decline, were surveyed in an orchard in Palermo province (Eastern Sicily, Italy). A Pleurostoma-like fungus was consistently isolated from symptomatic wood tissues. Based on morphology and phylogenetic analysis of ITS and tub2 sequences, the fungus was identified as Pleurostoma richardsiae. A pathogenicity test was conducted by inoculating stems of 2-year-old mango seedlings with mycelium plugs and conidium suspensions of a representative isolate. Two months after inoculation, necrotic lesions were observed around the inoculation points, and P. richardsiae was reisolated from the necrotic tissues. This is the first report of P. richardsiae causing dieback and decline of mango trees.

Characterization and Pathogenicity of Botryosphaeriaceae Species Associated with Gummosis, Dieback, Trunk and Branch Cankers of Almond Trees in Türkiye

Members of Botryosphaeriaceae family with 25 genera and several species are spread over a wide range of lands and climates worldwide. They cause gummosis, decline, dieback and blight on many woody plants. The purpose of present study was to diagnose the pathogens linked to the aforementioned symptoms on almond trees in seven orchards of Yozgat province (Türkiye) with a DSb type climate (Hot humid continental - Köppen Geiger system of climatic classification).These trees indicated and displayed dieback, gummosis trunk and branch canker symptoms. They were identified by cultural and morphological characteristics, and compared by phylogenetic sequencing of the ITS regions, EF-1α and β-tubulin genes with those of other species in GenBank (NCBI). Diplodia seriata, Lasiodiplodia theobromae, Neofusicoccum parvum and Botryosphaeria dothidea were identified in 72 isolates based on the colony and conidial characteristics. Successful pathogenicity tests were carried out on two-year-old almond seedlings of cv: Ferredual using Koch’s postulates. The results validated the identification According to available literature on the subject, identification of B. dothidea was done for the first time on almond trees in Türkiye. Accurate identification, prevalence and incidence of the pathogens are crucial for developing effective disease management strategies to arrest disease outbreaks in Türkiye.

Cultural, Morphological and Pathogenic Variability in the Colletotrichum capsici Isolates, Inciting Anthracnose and Fruit rot of Chilli (Capsicum annuum L.) in different Agro-Climatic Zones of Kerala, India

Aims: To study the cultural, morphological and pathogenic variability existing among the pathogen isolates of Colletotrichum capsici, inciting anthracnose and fruit rot of chilli in Kerala. Study Design: Completely randomized design (CRD). Place and Duration of Study: Department of Plant Pathology, College of Agriculture (Kerala Agricultural University), Vellayani, Thiruvananthapuram during 2018-2019. Methodology: A survey was conducted in the five agro-climatic zones (ACZs) of Kerala viz., Northern zone, Central zone, Special problem zone, High range zone and Southern zone to assess the incidence and severity of anthracnose disease. The various symptoms in the surveyed locations were also noted. Pathogen isolates were grown in PDA medium in the laboratory and colony characters (colour and mycelial growth rate of pathogen colonies) were observed. Also, the morphological characters viz., mycelial width, shape and size of conidia, acervular diameter, number and size of setae, appressorial size were recorded. Results: Maximum anthracnose incidence of 90 per cent and severity of 52.60 per cent were noticed at Regional Agricultural Research Station (RARS), Pilicode (Northern zone). Characteristic leaf spot, fruit rot and die-back symptoms were observed in the survey locations. Pathogen colonies showed different shades of white, off-white to grey turning brown or black with regular or irregular margins and concentric rings of black acervuli in the potato dextrose agar (PDA) medium. The diameter of growth of C. capsici isolates ranged from 7.20 to 8.60 cm at 7 days after incubation. All the C. capsici isolates initiated hyaline mycelium having a width of 1.73 - 2.36 µm and produced numerous sickle shaped conidia with a size range of 19.42 - 20.46 µm × 2.16 - 3.09 µm. Black coloured, circular or ellipsoidal acervuli (122.14 - 189.08 µm dia.) had 20 – 46 setae of length 74.13 to 107.30 µm. Also, brown or black appressoria of size 8.64 - 12.64 µm × 5.54 - 7.84 µm were formed in slide culture. Cc3 from College of Agriculture, Vellanikkara was obtained as the most virulent isolate producing a lesion size of 1.13 cm and PDI of 45.33 in the artificially inoculated chilli fruits (var. Vellayani Athulya) at 5 days after inoculation. Conclusion: All the nine C. capsici isolates exhibited variability in their cultural, morphological and pathogenic characteristics. Variability in phytopathogenic fungi helps in identifying novel and sustainable control methods to tackle the diseases incited by them. Understanding pathogen variability provides new insights for developing effective disease management methods. This will help in promoting sustainable agriculture and preventing severe crop losses.

First report of Ralstonia pseudosolanacearum causing bacterial wilt on Rosa sp. in Greece.

In March 2021, a sample of nine-month-old, non-grafted, diseased rose (Rosa sp.) plants was sent by a grower to the Benaki Phytopathological Institute for examination. The plants exhibited symptoms of dieback with black necrosis of pruned shoots, brown discoloration of shoot and root vascular tissues, and whitish slime exudation on cutting wounds of the shoots. The symptoms resembled those caused by Ralstonia pseudosolanacearum (Tjou-Tam-Sin et al. 2016). According to the sample's information sheet, the sample had been collected in a commercial greenhouse rose crop for cut flowers with a 10% disease incidence in the area of Troizinia-Methana (Regional Unit of Islands, Greece). Microscopic examination of symptomatic shoot and root vascular tissues revealed masses of bacterial cells streaming out of them. Sections of symptomatic tissues were suspended in water and in the resulting suspension, bacteria of the R. solanacearum species complex (RSSC) were detected by an indirect immunofluorescence (IF) assay using polyclonal antibodies (Plant Research International, the Netherlands) and a qPCR assay (RS-I-F/RS-II-R primers, RSP-55T probe) (Vreeburg et al. 2016). Furthermore, colonies with typical characteristics of RSSC were isolated from vascular tissues of shoots and roots on non-selective (NA) and semi-selective (mSMSA) media (EPPO 2022), and their identification as RSSC was confirmed by the above-mentioned IF and qPCR assays. Also, the isolates were assigned to: i) biovar 3, based on their ability to metabolize three disaccharides (maltose, lactose, D(+) cellobiose) and three hexose alcohols (mannitol, sorbitol, dulcitol) producing acid (EU 2006) and ii) phylotype I, by multiplex conventional PCR (Opina et al. 1997; Fegan and Prior 2005). A representative isolate was selected for sequencing part of the genes: 16S rDNA (1464bp), mutS (729bp) and egl (795bp) with GenBank Accession Nos. OR102443, OR683617 and OR702781, respectively. Blast analysis of these sequences showed 100% identity with those of various RSSC strains (e.g. GenBank Ac. Nos. CP025741.1, CP021762.1, MF141029.1, respectively). The obtained egl sequence conforms with the characteristics of phylotype I based on the DNA barcoding tool (EPPO 2021) and is 100% identical to that of the Dutch strain PD7216 (MF141029.1) reported to be sequevar I-33 (Bergsma-Vlami et al. 2018). The pathogenicity of two isolates was tested by inoculating: i) tomato seedlings (cv. 'Belladona') at their stem between the cotyledons and the first true leaf (EU 2006) and b) rose plants (cv. 'Aqua' and 'Papa Meilland') at their shoot base (Tjou-Tam-Sin et al. 2016), with bacterial suspensions in water (108 cfu/ml). The inoculated plants were maintained at a day/night temperature about 28/20°C with tomato plants exhibiting leaf wilting (7-17 dpi) and rose plants exhibiting chlorosis and necrosis of leaves (17 dpi). The pathogen was re-isolated on mSMSA from both artificially infected plant species and identified by the IF assay described above, thus fulfilling Koch's postulates. This is the first diagnosis in Greece of: i) rose plants infected by a Ralstonia species and ii) a crop infected by R. solanacearum phylotype I that corresponds to the R. pseudosolanacearum species (EPPO 2022). Official phytosanitary measures imposed in the affected area include an annual survey of rose crops for the presence of this pathogen, aiming at an early detection and prevention of its spread in such a highly valued ornamental crop.

Identifying the optimal temperature and water activity conditions of phytopathogenic fungi recovered from Al-Baha province

AbstractFungi are responsible for many plant infections. Indeed, fungi are responsible for most plant spoilage that occurs in the field or in storage, causing symptoms of dieback, leaf spot, rot, rust and wilt. A more serious issue is that fungi produce mycotoxins, which can contaminate crops. Samples of leaf, fruit and root tissues were collected from sick crops, pre- and post-harvest within Al-Baha region. Using molecular markers, twenty fungal isolates were identified that represent eight genera and thirteen species. The most common species were Penicillium spp. (45%), followed by Aspergillus spp. (25%). The remaining 30% were comprised of multiple species (Alternaria alternata, Fusarium oxysporum, Geotrichum candidum, Lasiodiplodia theobromae, Megasporoporia minor and Rhizopus arrhizu). The majority of these isolates are recognised as spoilers of crops and producers of mycotoxins. The study evaluated which temperatures optimised fungal growth. Half of the isolates thrived best at 25 °C; six isolates favoured 30 °C, whilst four isolates grew best at 20 °C. The study also evaluated the optimal water activity (aw) for fungal growth, at four points (0.995, 0.99, 0.90 and 0.85 aw). The isolates were categorised as being either xerophilic, mesophilic or hydrophilic. The majority of isolates (70%) were xerophilic (Aspergillus and Penicillium spp.), 10% were mesophilic (Alternaria alternata and Rhizopus oryzae spp.) and 20% were hydrophilic (Lasiodiplodia theobromae, Geotrichum candidum, Megasporoporia minor and Fusarium oxysporum spp). The information derived from this study will assist in building effective strategies to control these pathogens, minimising crops losses and mycotoxin contamination.