Dieback Symptoms Research Articles

First Report of Root Rot Caused by Phytophthora acerina on Metasequoia glyptostroboides in China.

Metasequoia glyptostroboides Hu & W. C. Cheng (Taxodiaceae), commonly called the Chinese redwood or dawn redwood, is a well-known "living fossil" and rare relict plant species endemic to China, which has been successfully cultivated throughout the world (Ma 2007). In July to September 2020, trees of Chinese redwood which were more than thirty years-old, showed symptoms of decline and death associated with branch dieback, root and collar rot (Fig. 1) in Yangtze River shelter-forests of Jiangling County in Hubei Province, China (112°15'19″E, 30°11'56″N; 40m). Diseased roots and rhizosphere soils were collected in September 2020 and April 2021. Using the baiting method, a homothallic Phytophthora sp. was recovered consistently from diseased roots and soil samples of Chinese redwood. All the isolates of this Phytophthora sp. formed similar colonies on V8 agar and corn meal agar (Fig. 2), and then three representative isolates (L4-5-4, L4-5-5 and L4-5-6) were randomly selected for morphological and molecular identification. In distilled water, semipapillate persistent sporangia were borne in simple sympodial branched sporangiophores. Sporangia were predominantly ovoid (Fig. 3a, d and f), but other shapes were observed including subglobose (Fig. 3b), limoniform (Fig. 3c) or distorted shapes (Fig. 3e), averaging 44.1 ± 7.7 µm (n=102) in length and 32.8 ± 5.2 µm (n=102) in width, with narrow exit pores of 8.0 ± 1.4 µm (n=93) and a length/breadth ratio of 1.3 ± 0.10 (n=102). Chlamydospores were not observed. Oogonia were globose or subglobose, 20.51 to 40.15 µm (av. 33.1 ± 3.9 µm) (n=119) in diameter, with smooth walls and paragynous antheridium (Fig. 3g-i). Oospores were globose or subglobose in elongated oogonia with medium wall thickness of 1.9 ± 0.5 µm (n=36), aplerotic or plerotic and 16.9 to 32.6 µm in diameter (av. 26.6 ± 3.8 µm) (n=40). According to the above morphological characteristics, this Phytophthora sp. was placed in Waterhouse's (1963) group III. The sequences of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA of each isolate (GenBank Accession No. OK087320, OK087321 and OK087322) was 760 bp and had identity of 99.84% with three P. acerina isolates (JX951285, JX951291 and JX951296), while the 800 bp β-tubulin (BTUB) sequences (OK140540, OK140541 and OK140542) showed 99.97% homology to the sequence of P. acerina (KC201283) (Ginetti, Moricca and Squires 2014) (Table 1). The ML phylogenetic trees were established by comparing ITS and BTUB sequences of three Phytophthora strains (L4-5-4, L4-5-5 and L4-5-6) with reference sequences of isolates of Phytophthora in ITS and BTUB in GenBank (Fig. 4-5). Based on the morphological and molecular characteristics, the strains were identified as namely P. acerina. In addition, pathogenicity assays were performed with one of the three strains (L4-5-4) on M. glyptostroboides using both one year old and three years old seedlings. Inoculum was prepared by subculturing agar plugs from edges of CMA cultures into V8 medium plates, incubating at 20 ℃ in darkness for 10 days. Six seedlings planted in pots filled with sterilized soil were inoculated by mycelium plug at root collar and stem wounded by a 8 mm diameter puncher. Six control seedlings were inoculated in the same manner as above, and sterile agar plugs were used. After 35 days, inoculated seedlings all had necrotic lesions at the inoculation sites, and some seedlings had the symptoms of foliage blight and dieback, whereas control seedlings remained healthy (Fig. 6). The number of fibrous roots after inoculation was significantly less than the control, and the roots of inoculated seedlings blackened or even rotted, while there were no obvious symptoms in the control (Fig. 7). Phytophthora isolates recovered from the symptomatic tissues of artificially inoculated plants were identical to isolate L4-5-4 in morphological characters and ITS sequencing. This is the first report of P. acerina causing root rot on the Chinese redwood in China. As only the seedlings were inoculated, further research is needed to address the epidemiology and pathogenicity of P. acerina to adult trees of Chinese red wood.

Gaziantep bağ alanlarındaki endofitik funguslar ve bunların fungal gövde patojenleriyle antagonistik ilişkileri

Son yıllarda Türkiye ve dünya bağlarında, asma gövde hastalıklarıyla sıkça karşılaşılmaktadır. Bu çalışmanın amaçları, Gaziantep bağlarında fungal gövde patojenlerini saptamak ve fungal endofitlerin asma fungal gövde patojenleriyle antagonistik ilişkilerini araştırmaktır. Sörvey çalışması İslahiye, Nurdağı ve Şahinbey ilçelerinde bulunan 51 bağda, Eylül 2020’de yürütülmüş, hastalık çıkışı ve yaygınlığı ortaya konmuştur. Patojenik ve endofitik türler arasındaki antagonistik ilişkiler PDA (patates dekstroz agar) besi yerinde ikili kültür testiyle belirlenmiştir. Sonuçlara göre, incelenen tüm bağlarda lokal kuruma, geriye doğru ölüm ve apopleksi belirtilerine rastlanmış, bölgedeki ortalama hastalık sıklığı %1.2 olarak hesaplanmıştır. Tanı çalışmaları ve patojenisite testleri bölgedeki bağların asma gövde hastalıklarıyla ilişkili Biscogniauxia, Botryosphaeria, Cytospora, Diatrype, Diplodia, Eutypa, Neoscytalidium, Phaeoacremonium, Seimatosporium ve Kalmusia türleri ile enfekteli olduğunu göstermiştir. İkili kültür testlerinde, Penicillium fructuariae-cellae’nın ortalama 9.04 mm’lik inhibisyon zonuyla çoğu asma gövde patojenlerine antagonistik olduğu tespit edilmiştir. Bu sonuçlar, asma gövde hastalıklarının mücadelesinde yeni fungal biyokontrol etmenlerini geliştirmek için endofitik Penicillium’lara daha fazla dikkat edilmesi gerektiğini göstermektedir.

Neoscytalidium novaehollandiae causes dieback on Pinus eldarica and its potential for infection of urban forest trees

Neoscytalidium novaehollandiae is one of the most important pathogens on woody plants which has increasingly been reported as a pathogen in different hosts in recent years. The pine trees are widely cultured in many cities of Iran. In recent years, dieback symptoms were observed on Pinus eldarica trees in Tehran and Qazvin provinces. The aim of this study was to investigate the dieback causal agent on P. eldarica trees in Iran. The branches and cones of P. eldarica trees were sampled for fungal isolation. The morphological and molecular characterizations (ITS, LSU, and TEF1-α regions) identified N. novaehollandiae as a dieback causal agent. This is the first report of N. novaehollandiae disease of P. eldarica trees in Iran. Furthermore, disease severity was assayed on 19 urban forest trees under three different temperature and relative humidity (RHs) regimes. C regime (29 °C and 15% RH) displayed more disease severity on detached branches than B (24 °C and 80% RH) and A (19 °C and 35% RH) ones. This study presents the host range of this pathogen, and showed that these potential hosts are prone to this pathogen under high temperature and low humidity which urban forest trees experienced in recent decades.

First Report of Diaporthe ambigua Associated with Dead Arm Disease on Grapevine in Chile.

Grapevine (Vitis vinifera L.) is one of the most important fruit crops in Chile based on economic value. Phaeomoniella chlamydospora and Botryosphaeriaceae species have been reported as the major causal agents associated with dieback symptoms in Chile commercial vineyards (Díaz and Latorre 2014; Besoain, 2018; Larach et al. 2020). Recently Eutypa lata has been reported attacking Chilean vineyards with dieback symptoms (Lolas et al. 2020). In this study, two commercial cv. Cabernet Sauvignon vineyards, located in O'Higgins Region of Chile, showing dead cordons, dead spur with a grayish color, canker, and vascular necrosis were sampled in fall 2018, with a high incidence of symptoms was observed. Four symptomatic wood samples were analyzed from these vineyards. Pieces of wood (<1 cm2) were taken from the advance zone of the canker lesions, disinfected with 70% ethanol, rinsed in sterile distilled water, dried, and transferred to two media in Petri plates, potato dextrose agar acidified with 0.5 ml of 96% lactic acid (APDA) and malt extract agar, and incubated for at least seven days at 24°C in darkness. From mycelium obtained from monosporic culture, two isolates were selected and morphologically identified as Diaporthe sp. To induce sporulation, these two isolates were grown in APDA under near-ultraviolet light (λ = 320 nm) at room temperature. After 30 days, the development of pycnidia was observed. Both Diaporthe sp. isolate presented alpha-conidia ellipsoidal with an obtuse apex, biguttulate (n=30) of 6.7 µm ± 0.33 µm x 3.3 µm ± 0.32 µm. No Beta-conidia or perhitecia were observed. DNA was extracted from the monosporic mycelium. The ribosomal internal transcribed spacer (ITS), β-tubulin (BT) gene, and elongation factor (EF) gene were amplified using ITS4/ITS5, Bt2a/Bt2b, and EF1-728F/EF1-986R primer pairs, respectively. PCR products were sequenced and identified as Diaporthe ambigua Nitschke (PUCV2140 and PUCV2141), showing 100% sequence identity with ITS MH864620.1, 99.8% with BT MG281142.1, and 100% with EF KC343738.1 sequences from D. ambigua. Sequences were deposited in GenBank (ITS: accession numbers MW301136, MW301137; BT: MW323445, MW323446 and EF: MW308305, MW308306). Two pathogenicity tests were performed with strains PUCV2140 and PUCV2141 using 2-year-old V.vinifera cv. Cabernet Sauvignon. In each test, three plants were used per isolate, considering one plant as an experimental unit. In the first test, a 5 mm mycelial plug from a 6-day-old APDA culture was inoculated using an oblique cut made in the bark with a sterile scalpel and done at the middle of the trunk. In the second test, the trial was done under the same described conditions previously, but in this case, one-year-old semi-lignified shoots were inoculated between two internodes, using mycelial plugs, one shoot for each plant. Injured plants but treated with sterile APDA plugs were used as controls. Plants were placed in natural conditions, and after three months from inoculations, plants showed a cortical canker and brown vascular lesions. Non-inoculated plants remained asymptomatic. The lengths of the cankers were 22.0 ± 1.8 mm and 10.5 ± 0.6 mm, after inoculations of the trunk and cane, respectively. The vascular lesions were 37.0 ± 3.3 and 18.0 ± 2.0 mm, in trunk and cane inoculations, respectively. D. ambigua was re-isolated and reidentified morphologically from the inoculated symptomatic plants, confirming Koch's postulate. Also, the plants inoculated on the trunk showed premature leaf drop. To our knowledge, this is the first report of D. ambigua associated with dieback affecting grapevines in Chile. Previous D. ambigua was reported causing fruit rots (Auger et al. 2013; Díaz et al. 2017) and cordon dieback in kiwifruit (Díaz and Latorre, 2018), and stem canker and dieback in blueberry (Elfar et al. 2013) in Chile. This study reports a new species of fungi for Chile associated with the dead arm in vineyards. D. ambigua is a pathogen in essential crops in our country. Therefore, it is important to study its prevalence in the future.

Investigations on Fungi Isolated from Apple Trees with Die-Back Symptoms from Basilicata Region (Southern Italy).

Val d’Agri is an important orchard area located in the Basilicata Region (Southern Italy). A phenomenon affecting cv. “Golden Delicious” apples which lead to tree death has been observed in the past several years in this area. This phenomenon has already been detected in about 20 hectares and is rapidly expanding. The symptoms observed were “scaly bark” and extensive cankers, mainly located in the lower part of the trunk, associated with wood decay. Dead plants ranged from 20% to 80% and, in many cases, trees were removed by farmers. In order to identify the causes of this phenomenon, investigations were started in autumn/winter 2019. In order to determine the possible causal agents, fungal and bacterial isolations, from symptomatic tissues, were performed in laboratory. Bacterial isolations gave negative results, whereas pure fungal cultures (PFCs) were obtained after 3–4 passages on potato dextrose agar (PDA) media. Genetic material was extracted from each PFC and amplified by PCR using three pairs of primers: ITS5/4, Bt2a/Bt2b and ACT-512F/ACT-783R. The amplicons were directly sequenced, and nucleotide sequences were compared with those already present in the NCBI GenBank nucleotide database. All isolated fungi were identified based on morphological features and multilocus molecular analyses. Neofusicoccum parvum, Diaporthe eres and Trametes versicolor were most frequently isolated, while Pestalotiopsis funerea, Phomopsis spp. and Diaporthe foeniculina were less frequently isolated. All nucleotide sequences obtained in this study have been deposited into the EMBL database. Pathogenicity tests showed that N. parvum was the most pathogenic and aggressive fungus, while Phomopsis sp. was demonstrated to be the less virulent one. All the investigated fungi were repeatedly reisolated from artificially inoculated twigs of 2-year-old apple trees, cv. “Golden Delicious”, and subsequently morphologically and molecularly identified. The role played by the above-mentioned fungi in the alterations observed in field is also discussed.

First Report of Coffee Canker Disease Caused by Ceratocystis fimbriata in China.

Coffee (Coffea arabica L.) is one of the most important agricultural commodities in the world market. As an important cash crop in China, coffee is cultivated mainly in Yunnan and Hainan provinces. During October 2013 and September 2020, coffee trees showing typical dieback and wilt symptoms were found in Nanping town (N 22° 38', E 101° 0'), Pu'er, and Puwen town (N 22° 32', E 101° 4'), Xishuangbanna in Yunnan province, China. Symptomatic trees initially exhibited yellowing of foliage, expanding in size along the leaf margin, then became blighted and dry, and the internal xylem in main stem discolored brown to black. Infected trees eventually developed dieback and wilt. Disease incidence ranged from 10% to 22% and 25% to 40% of crown symptoms in the affected coffee trees. In extreme cases, 50% out of 380 trees were affected. The stems of coffee trees with typical symptoms were collected, and then the diseased tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, fungal mycelium was observed growing from the tissue. Three isolates (C3-1, C3-2, and C3-2-1) were obtained by picking spore masses from the apices of perithecia and transferring them to malt extract agar (MEA) medium and incubated at 25°C for 10 days to observe the cultural features. In culture, colonies reaching 65 mm within 10 days, mycelium initially white, then becoming light blue-green. After 6 days of formation, perithecia were black, globose (123.8 - 173.4 μm × 138.2 - 180.6 μm), and showed a long black neck (414.2 - 650.0 μm). Ascospores with outer cell wall forming a brim, hat-shaped, accumulating in a mucilaginous mass at the tips of ostiolar hyphae (4.3 μm × 6.0 μm). Cylindrical endoconidia (14.1 - 45.2 μm × 3.5 - 5.7 μm) were hyaline. Chain of barrel-shaped conidia (6.6 - 10.2 μm × 6.8 - 8.8 μm) were found. Aleuroconidia (10.8 - 16.9 μm × 9.1 - 13.0 μm) were olive-brown, ovoid or obpyriform, and smooth. Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata Ellis & Halst. (Engelbrecht and Harrington 2005). The three isolates were used for molecular identification, and their genomic DNA was extracted using the chelex-100 method (Xu et al. 2020). The internal transcribed spacer (ITS) region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of the ITS sequence data (GenBank accessions KY580836, KJ511480, and KJ511479) showed that the isolates were 100% homologous to isolates of C. fimbriata from Punica granatum, Camellia sinensis, and Cucumis sativus in China (GenBank accessions KY580891, KY580870, and MH535909, respectively) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis was performed using MEGA 6.06 based on the ITS sequences. The three isolates were clustered on the same clade with other C. fimbriata isolates with a high bootstrap value (90%). Therefore, the fungus was identified as C. fimbriata based on both morphological and molecular characteristics. Pathogenicity of the three isolates was tested by inoculating one-year-old pot grown coffee seedlings (C. arabica) through drenching the loams with 30 ml spore suspension (1 × 106 spores/ml). Control plants were inoculated with 30 ml of sterile distilled water. The trees were kept in a controlled greenhouse at 25°C and watered weekly. One month after inoculation, all inoculated plants produced typical dieback and wilt symptoms, whereas the control trees showed no symptoms. The same fungus was isolated from the inoculated trees on PDA and identified as C. fimbriata according to the methods described above, and no fungal growth was observed in the controls, thus fulfilling the Koch's postulates. Coffee canker disease caused by C. fimbriata has been reported in Indonesia and Colombia (Marin et al. 2003). To our knowledge, this is the first report of C. fimbriata causing canker disease of coffee trees in China.

First Report of a New Disease Caused by Fusarium tricinctum on Apple Tree in China.

First Report of a New Disease Caused by Fusarium tricinctum on Apple Tree in China.

Fungal endophytes in Fraxinus excelsior petioles and their in vitro antagonistic potential against the ash dieback pathogen Hymenoscyphus fraxineus.

Fungal endophytes were isolated from 250 asymptomatic leaf petioles of Fraxinus excelsior collected from trees showing symptoms of ash dieback in five forest sites in southern Poland. Fungal isolations yielded 1646 colonies representing 97 taxa, including 92 Ascomycota and 5 Basidiomycota species. The most common Ascomycota comprised Nemania serpens (38.0 % of colonized petioles), Diaporthe eres (33.6 %), Venturia fraxini (26.4 %), Diaporthe sp. 1 (20.4 %), Alternaria sp. 1 (14.8 %), Colletotrichum acutatum (14.8 %), Nemania diffusa (14.0 %), Colletotrichum gloeosporioides (12.4 %) and Colletotrichum sp. (12.4 %). The occurrence of all these taxa except Alternaria sp. 1 was significantly different between the studied forest sites. Two yeast species, Vishniacozyma foliicola (4.8 %) and Cystobasidium pinicola (2.8 %), dominated among the Basidiomycota endophytes detected. All the fungal endophytes were tested in dual culture antagonistic assays against two strains of Hymenoscyphus fraxineus, resulting in the development of four interaction types. The interactions included the physical contact of co-partners' mycelia (41.8 %), development of an inhibition zone (47.4 %), growth of endophyte mycelia over H. fraxineus colonies (9.3 %) and growth of H. fraxineus mycelia over endophyte colonies (1.5 %). The strongest antibiotic activity against H. fraxineus, measured by the width of the inhibition zone, was observed for Cytospora pruinosa, Fusarium lateritium, Phoma sp. 2, Pleosporales sp. 2 and Thielavia basicola. A variety of morphophysiological deformations of H. fraxineus hyphae were observed under endophyte pressure: spiral twist of the hyphae, formation of cytoplasmic extrusions, development of torulose hyphae and excessive lateral branching of the hyphae. The strongest antagonistic effects, coupled with the potential to overgrow H. fraxineus colonies, was shown by Clonostachys rosea, Nemania diffusa, N. serpens, Peniophora cinerea, Rosellinia corticium and Xylaria polymorpha. Some of these species were able to attack H. fraxineus hyphae in a mycoparasitic manner. The antagonistic activities included the physical penetration of H. fraxineus hyphae, dissolution of hyphal cell walls, disappearance of pigmentation, disintegration of hyphae and degradation of other fungal structures. In contrast, one of the most commonly detected endophytes in ash leaves, Venturia fraxini, did not show in vitro antagonistic potential against H. fraxineus. Finally, we discuss the potential of the detected fungal endophytes to combat H. fraxineus invasion, the cause of ash decline in Europe.

Nutrient Deficiency Induces Branch and Shoot Dieback in Robusta Coffee

In southern Sumatra, Robusta coffee is grown mainly in mountain regions, undulating and steep lands, and marginal soils. Management of soil nutrients is a key issue to maintain sustainable coffee production. This survey was conducted at the field of Farmer Training Center (FTC), at Pulau Panggung Village, Semendo Darat Laut Sub-district, Muara Enim District of South Sumatra Province, Indonesia. The trees of seven coffee clones were randomly sampled. The soil of 0-20 cm depth and full-grown leaves from middle branches was compositely sampled. The results of soil analysis and visual symptoms showed a deficiency of macro nutrient including N, P, K, and Mg that coincident with a high incidence of dieback symptoms. We observed that branch dieback incidence was 62-100% with severity score 1.0-2.5 and shoots dieback incidence was 15-50% with severity score 1.0-3.0. Local coffee clone showed a better tolerance on branch and shoots dieback and higher yield potential based on number of fruits and pinhead fruit per bunch. The results of observation on leaf symptoms of nutrient deficiency, slow plant growth, dieback incidence, plant death, and low plant population showed as a failure of coffee establishment induced by low soil fertility and poor plant management.

Bademde Kanser ve Geriye Doğru Ölüm Etmeni Neoscytalidium novaehollandiae’nın Tanılanması, Patojenisitesi ve Bazı Fungisitlerin In Vitro Etkinlikleri

The objective of the present study is to identify the causal agents associated with cancer and dieback symptoms in almond production areas in Batman's Binatlı village, to determine the pathogenicity of the causal agent, and to determine the efficacy of some fungicides on the pathogen in vitro. As a result of the morphological and molecular characterization of fungal isolates obtained from diseased plant tissues, the fungus was identified as Neoscytalidium novaehollandiae. In the pathogenicity test of 5 isolates of N. novaehollandiae on almond seedlings cv Ferragnes, AlNnov79 was determined as the most virulent isolate. As a result of the efficacy trial of five different fungicides on N. novaehollandiae isolate AlNov79, highly effective fungicides were determined as fluazinam (EC50;0.002 µl ml-1), thiophanate–methyl (EC50;0.3 µl ml-1), tebuconazole (EC50;0.4 µl ml-1), boscalid + pyraclostrobin (EC50;2.2 µl ml-1). Trifloxystrobin (EC50;19.5 µl ml-1) was found as the lowest effective fungicide.

Identity and pathogenicity of Botryosphaeriaceae and Diaporthaceae from Juglans regia in Chile

English walnut (Juglans regia) has become an important crop in Chile, representing 11.5% of the total area of fruit trees, surpassed only by grapevine. As the Chilean walnut industry rapidly expands, young orchards are at risk from the emergence of new fungal diseases. Botryosphaeriaceae and Diaporthaceae fungi have been recognized as main causes of wood diseases in walnut, with symptoms of dieback, canker, and blight. In winter 2017, samples were collected from different orchards in Valparaíso and Maule regions. Fungal isolates recovered were cultured, characterized morphologically, and identified using DNA sequence analyses. Three species (Neofusicoccum parvum, Diplodia mutila, Diplodia seriata) were characterized in Botryosphaeriaceae and two (Diaporthe cynaroidis, Diaporthe australafricana) in Diaporthaceae. Pathogenicity tests showed that N. parvum was the most aggressive species to walnut. This study confirmed the presence of pathogenic Botryosphaeriaceae and Diaporthaceae in J. regia that should be considered an increasing risk for the growing Chilean walnut industry.

First Report of Coniella granati Associated with Dieback of Rose (Rosa sp.) in India.

Rose (Rosa sp.) is an important floricultural crop largely cultivated in the Karnataka state of southern India. A field survey conducted in Devanahalli, Bangalore rural during October, 2019 revealed rose plants (cv. Arka Pride) showing die-back symptoms with a disease incidence of 7% in an area of 30 hectare rose field. Die-back was characterized by the development of necrotic lesions that started at the tip of shoots and progressed downwards leading to the death of young shoots. Pycnidia were found on young shoots showing necrotic lesions. Die-back affected shoots (n=10) were surface sterilized with 2% NaOCl for 2 min, rinsed thrice in sterile distilled water, and plated on potato dextrose agar (PDA) medium amended with chloramphenicol (40mg/L). The plates were incubated at 28±2 °C and pure cultures were obtained by hyphal tipping. Fungal colonies with white aerial mycelia along with pycnidia were observed on eight samples. Colony growth rate was at 4.2 ± 0.4 mm/day with an average colony diameter of 47.2 ± 3.8 mm after 7 days (12/12 h light and dark period). Pycnidia were solitary and globose. Conidia were hyaline, single celled ellipsoid to fusiform, measured 12.4 - 16.78 × 3.2 - 5.1 μm (n=50). Based on cultural and morphological features, the fungus was identified as Coniella granati (Van Niekerk et al., 2004; Mahadevakumar et al., 2019) (Figure S1). Further, two representative isolates (NLD-2021-1 & NLD-2021-2) were used for molecular identification based on barcoding of ITS-rDNA, LSU and tef1 regions amplified using primer pairs described in Alvarez et al., 2016. PCR amplified products were sequenced and the consensus sequence were analyzed through BLASTn search which showed ITS (NLD-2021-1: 611/611bp - MH860368 & NLD-2021-2: 573/573bp - KU147239), LSU (NLD-2021-1: 783/906bp - MH872113 & NLD-2021-2: 844/1172bp - KX833400) and tef1 (NLD-2021-1 & NLD-2021-2: 548/548bp -KX833682) sequences shared 100% sequence similarity with C. granati respectively. The nucleotide sequences were deposited in GenBank with the accession numbers ITS: MW898436, MW898437; LSU: MZ895475, MZ895476 and tef1 MW916275, MW916276 respectively. The phylogenetic tree constructed based on combined ITS-LSU-tef1 loci confirmed that the sequences shared a common clade with C. granati (Figure S2). Further, pathogenicity tests were conducted on conducted twice on healthy rose plants (Arka Pride; 60 days old after grafting) maintained in a greenhouse at 28±2°C and 70% relative humidity. Whole plant inoculation was followed by spraying the conidial suspension (10ml / plant) of C. granati (NLD-2021-1) (3 × 106 conidia/ml, amended with Tween20 at 2%) on 15 plants. A total of 10 healthy plants inoculated with sterile distilled water amended with Tween20 at 2% served as control. Inoculated plants were covered with polythene bags for 48 h to maintain relative humidity. Die-back symptoms were observed on young shoots after 15-18 days of post inoculation (DPI), and control plants remained disease free after 15 DPI. The pathogen was re-isolated on PDA from 10 diseased plants and identity was confirmed through morphological methods. Coniella granati is an important phytopathogen known to cause die-back and twig blight of pomegranate globally and other economically important crops (Cintora-Martinez et al. 2017). No reports are available on the association of C. granati with dieback of rose (Farr & Rossman, 2021), which should help in developing appropriate disease management strategies.

First Report of Diaporthe foeniculina Associated with Grapevine Trunk Diseases on Vitis vinifera in Cyprus.

In June 2017, three vineyards were surveyed in the regions of Droushia (30-year-old, cv Mavro), Ineia (50-year-old, cv Xynisteri), and Lemona (15-year-old, cv Carignan) at the province of Paphos, Cyprus, with dieback incidence of 22%, 32%, and 14%, respectively. More specifically, affected grapevines exhibited severe dieback symptoms in spur and cordon positions, related to perennial cankers and internal brown discoloration. Thirty symptomatic samples, were surface-sterilized (95% ethanol) and wood chips were plated on potato dextrose agar (PDA), amended with streptomycin (500 μg/ml) at 25 °C for 3-5 days. Based on colony morphology (white to creamy color, with sparse aerial mycelium) and conidia production, nine Diaporthe-like isolates were obtained. For species identification, the internal transcribed spacer (ITS) region and β-tubulin (BT) genes were amplified using the primer pairs ITS1/ITS4 and Bt2a/Bt2b, respectively (Úrbez-Torres et al. 2008). Sequences of the isolates P101b, P114c, and P289a revealed >99.8% homology to NCBI voucher specimens of Diaporthe foeniculina (Sacc.) Udayanga & Castl. (ITS: CBS111553, MH050434; ΒΤ: KY511368, KF778966), and were deposited in the GeneBank (ITS: MT735646, MT737289, MT737287; BT: MT903969, MT903970, MT903971). Thus, 8.3% of the collected isolates (3 of 36) were identified as D. foeniculina, while the rest Diaporthe-like isolates were identified as D. ampelina. D. foeniculina isolates were also transferred on 2% water agar with sterile pine needles under a 12h/12h near-ultraviolet, light/darkness regime, at 25 °C, to induce sporulation (Guarnaccia and Crous 2017). Two weeks later, microscopic observations revealed dark brown to black, globose to sub-globose, ostiolate pycnidia (n = 30) 291 to 897 μm (595 ± 173) x 192 to 655 μm (364 ± 113) containing hyaline, unbranched conidiophores, bearing alpha- and beta-conidia in the form of yellowish cirri. Alpha-conidia were aseptate, hyaline, ovate to ellipsoidal, ranging (n=100) from 5.6 to 9.9 μm (7.5 ± 0.8) x 1.9 to 3.3 μm (2.7 ± 0.3). Beta-conidia were abundant, aseptate, hyaline, filiform, slightly curved (n = 100) from 22.4 to 35.3 μm (28.1 ± 2.5) x 1.2 to 2.3 μm (1.6 ± 0.2) (Udayanga et al. 2014). Pathogenicity tests were conducted with isolates P101b and P289a under greenhouse conditions (24-32 ⁰C, 70% RH). Ten 1-year-old rooted canes cv Mavro were inoculated with 4 mm mycelium plugs from actively growing cultures into wounds made by drilling between two internodes at the middle of the trunk. The same number of cuttings were inoculated with sterile PDA plugs, sealed with Vaseline, and wrapped with parafilm, serving as controls. Seven months later, all inoculated cuttings developed brownish wood discolorations (average 39 ± 13 mm), similar to naturally infected plants. No symptoms were observed in the controls. Successful re-isolations were made only from the inoculated cuttings and confirmed by colony morphology. Previously, D. foeniculina (as D. neotheicola) has been reported as grapevine wood saprophyte (Úrbez-Torres et al. 2014). It has also been reported to cause shoot canker and dieback in numerous hosts, including almond, avocado, citrus, and sweet chestnut (Annesi et al. 2016; Guarnaccia and Crous 2017; Diogo et al. 2010; Mathioudakis et al. 2020). This is the first record of D. foeniculina associated with grapevine trunk diseases (GTDs) in Cyprus. However, its relative importance as the causal agent of GTDs remains to be further investigated.

Morphological and Molecular Characterization of Botryosphaeriaceae Species Associated with Dieback and Gummosis on Plum Trees in Turkey

Plum (Prunus salicina) is among the most important and common fruit species in Turkey. Worldwide, Botryosphaeriaceae family, including significant plant pathogens causes disease in several woody plants including plums. Botryosphaeriaceae-induced dieback and gummosis symptoms have recently been encountered in plum orchards in Adana and Mersin provinces of Turkey. In the present study, morphological and molecular characterization of Botryosphaeriaceae isolates were performed and the obtained isolates were identified as Diplodia seriata, Neofusicoccum parvum and Lasiodiplodia pseudotheobromae. Pathogenicity tests revealed the virulence of all three species. To the best of our knowledge, this is the first report of D. seriata, N. parvum and L. pseudotheobromae causing disease on plums in Turkey.

Characterization and Pathogenicity of Diplodia, Lasiodiplodia, and Neofusicoccum Species Causing Botryosphaeria Canker and Dieback of Apple Trees in Central Chile.

In recent years, the number of apple trees affected by Botryosphaeria cankers and dieback has considerably increased in central Chile. This study aimed to identify the species of Botryosphaeriaceae associated with canker and dieback symptoms, estimate disease incidence and distributions, and study their pathogenicity and virulence on apple and other fruit crops. A field survey of 34 commercial orchards of apple (7 to 30 years old) was conducted in 16 localities, obtaining 270 symptomatic branch and trunk samples in 2017 and 2018 growing seasons. The incidence of Botryosphaeria canker and dieback ranged between 5 and 40%, and a total of 255 isolates of Botryosphaeriaceae spp. were obtained from 238 cankers. Morphological identification along with phylogenetic studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the translation elongation factor 1-α (tef1-α), and part of the β-tubulin (tub2) genes allowed us to identify Diplodia mutila (n = 49 isolates), D. seriata (n = 136 isolates), Lasiodiplodia theobromae (n = 16 isolates), and Neofusicoccum arbuti (n = 54 isolates). L. theobromae was isolated mainly from apple dieback from northern localities. All pathogens tested were pathogenic, causing canker and dieback symptoms on lignified twigs of apple, pear, walnut, and green grapevine shoots in the field. Isolates of N. arbuti were the most virulent, reproducing more severe cankers on the lignified tissues inoculated. This study reports, for the first time, D. mutila and L. theobromae associated with Botryosphaeria canker and dieback in Chile, and it is the first description of N. arbuti causing apple dieback worldwide.

Occurrence and richness of arbuscular mycorrizal fungi in vineyards with grapevine decline and dieback symptoms

ABSTRACT: This research identified arbuscular mycorrhizal fungi (AMF) in rhizosphere soil of grapevines with Grapevine Death and Decline symptoms (GDD) or asymptomatic healthy (H) plants, and characterized the relationship of AMF communities with soil chemical attributes. The AMF spore number ranged from 287 to 432 spores 50 cm-3 in soil with GDD plants, and from 357 to 464 spores 50 cm-3 in H plants, with no differences among vineyards or between GDD and H plants within each vineyard. We detected 42 species and 17 genera, and most taxa belonged to Acaulosporaceae or Glomeraceae. Claroideoglomus etunicatum, Funneliformis mosseae, and Archaeospora trappei were the most frequent species in all vineyards. Soil chemical attributes were not determinant for the occurrence of most fungal species; although, Entrophospora infrequens, Diversispora sp1 and Diversispora sp2 were associated with a vineyard having high soil copper. Vineyards harbor highly diverse AMF communities, which are determined by location.

First report of Diplodia seriata, D. mutila, and Dothiorella omnivora associated with apple cankers and dieback in Rio Negro, Argentina.

Apple (Malus domestica Borkh.) is an important fruit crop in Río Negro, Argentina. In recent years, the frequency of canker and dieback symptoms have increased affecting different apple cultivars. In September 2014, a higher occurrence of cankers (29%) and dieback (9%) was observed in a commercial orchard of 10-year-old apple trees (n=210) cv 'Ital Red' in General Roca, Río Negro, Argentina (39°2'36.73"S - 67°32'44.55"W). Symptoms initially appeared as necrotic bark lesions on tree trunks and branches often associated with pruning wounds. Superficially, papyrus detachment of the bark and cracked bark were observed on the affected area. When the bark was removed, the diseased wood showed dark brown color. Cross sections of diseased branches revealed necrotic lesions that progress to branch death. Samples were collected from different symptomatic trees (n=30) and were superficially disinfected with 70% ethanol. Internal tissues (0.5 cm2) were excised from the advance margin of the necrotic lesions, plated on potato dextrose agar (PDA), and incubated at 22°C. Pycnidia were induced on sterilised pine needles overlaid on 2% water agar under near-UV light. Optimum temperature of culture growth on PDA was studied. According to their morpho-cultural characteristics, three different morphotypes were identified. The first, showed optimum growth at 30°C, had moderately dense white aerial mycelia and turned dark gray after 7 d. Conidia were ovoid, mostly aseptate, 20.8-25.6 × 8-11.4 µm (n=50) and hyaline to brown. The second, exhibited optimum growth from 25 to 30°C, was white to gray, with sparse to moderate aerial mycelium that turned dark olive green. Conidia were ovoid, 1-septate, 17.6-22.4 × 8.1-11.2 µm (n=50) and brown. Finally, the third showed optimum growth at 25°C, mycelium was grey to dark olive green. Conidia were oblong to ovoid with both ends rounded, aseptate and 1-septate at maturity, 20.8-24 × 11.2-14.4 µm (n=50), hyaline turned brown. Genomic DNA was extracted from one representative isolate of each morphotype and the ITS and EF1-α loci were amplified with the primer sets ITS1/ITS4 (White et al., 1990) and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. The nucleotide sequences indicated ≥99% identity to D. seriata (CBS 114796 and CBS 112555), D. mutila (CBS 302.36 and CBS 112553), and D. juglandis (CBS 188.87), reclassified as Dothiorella omnivora (Linaldeddu et al., 2016), for both DNA regions. The sequences were deposited in the GenBank database (MW596418, MW598375; MH665432, MK955889; MH665413, MK937229). To confirm pathogenicity, healthy 1-year-old twigs of adult apple trees were pruned and wounds of attached twigs immediately inoculated with 20 μL of conidial suspension (103 conidia.mL-1, n=9 per isolate) or sterile distilled water (control, n=9), and wrapped with Parafilm. The experimental design was randomized, and the experiment was repeated once. After 90 d, the area of lesion on all twigs inoculated was determined. D. mutila and Do. omnivora produced mean canker areas (65 and 73 mm2, respectively) significantly larger (p < 0.005) than D. seriata (48 mm2). No lesion occurred in the negative controls. Fulfilling Koch's postulates, fungi were reisolated from all inoculated twigs and no fungus was recovered from controls. To our knowledge, this is the first report of D. seriata, D. mutila, and Do. omnivora associated with apple canker and dieback in Argentina, which shows the need of study the role of these fungi in orchard health.

Screening of Existing and New Boxwood Cultivars for Disease Resistance and In Vitro Fungicide Screening to Manage Boxwood Dieback Caused by Colletotrichum theobromicola

Boxwood (Buxus spp. L.) is one of the most common and widely planted perennial ornamentals in both home gardens and commercial landscapes. Although considered robust, boxwood is susceptible to several plant pathogens. Boxwood dieback, a recently reported fungal disease caused by Colletotrichum theobromicola, has been spreading rapidly within the United States. Symptoms of boxwood dieback are difficult to distinguish visually in affected plants due to the symptom resemblance to other known biotic and abiotic diseases of boxwood. Previous studies have shown that it may take 2 to 3 months for the symptoms to appear under greenhouse conditions. Although a molecular method has been designed for early, rapid, and accurate pathogen detection, boxwood breeders, nursery growers, and landscape professionals are concerned about the lack of effective management practices. Therefore, the primary objectives of this study were to devise effective disease management strategies by screening cultivars to determine their susceptibility to boxwood dieback and screening various fungicides to determine their effectiveness in managing the disease. Host range studies were conducted by screening a wide variety of boxwood cultivars under greenhouse conditions. The boxwood cultivar ‘Little Missy’ showed much-delayed symptom expression compared with the rest of the cultivars. None of the 11 cultivars were found to be resistant to boxwood dieback. In vitro screening of nine fungicide treatments was conducted to determine inhibition of mycelial growth and spore germination for eight isolates of C. theobromicola collected from eight states in the United States. Of the nine fungicide treatments, difenoconazole + pydiflumetofen showed maximum inhibition of mycelial growth and spore germination at 1 ppm active ingredient, followed by fluxapyroxad + pyraclostrobin, and pyraclostrobin + boscalid at 5 ppm active ingredient. Azoxystrobin + benzovindiflupyr significantly inhibited mycelial growth at 1 ppm but reduced spore germination only at 10 ppm active ingredient. This study provides boxwood industry professionals with critical information pertaining to host susceptibility and fungicide efficacy that may be applied to effectively mitigate boxwood dieback and reduce its further spread.

Characterization and pathogenicity of Diplodia seriata causing branch canker on Pinus pinea in Tunisia

Several species of Botryosphaeriaceae family are among the most aggressive pathogens associated with botryosphaeria dieback of agricultural and forestry trees. Particularly, Diplodia spp. having a cosmopolitan distribution, are well-known as virulent of woody plant hosts including Pinus spp. In recent years, symptoms of canopy wilt, branch dieback, necrosis, and trunk cankers have been noticed on Pinus pinea trees in Tunisian forests. However, this has been less welldocumented in North Africa and especially in Tunisia. The aim of this study is to characterize the causal agent of P. pinea dieback in northeastern Tunisian forest. A collection of thirty-eight isolates obtained from symptomatic branches of P. pinea trees was identified as Diplodia seriata by means of morphological characteristics, and phylogenetic analysis of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA. A pathogenicity test was conducted on 3-years-old P. pinea seedlings, confirmed the virulence of the fungus. To the best of our knowledge, this is the first record of D. seriata associated with branch canker on P. pinea in Tunisia.

Morphological and Phylogenetic Resolution of Diplodia neojuniperi Emerging Diplodia Top Dieback of Pinus thunbergii Parl. in China

In Bazhong City, Sichuan Province, China, top dieback symptoms were found on many pine trees (Pinus thunbergii Parl). The tips of old needles first turned grayish-green and then developed into brown bands in the field. Phylogenetic analysis of concatenated ITS and EF1-α indicated the pathogen of this dieback disease as Diplodia neojuniperi. Additionally, effects of temperature, pH and medium on the mycelial growth were also characterized. The most favorable temperature and pH level for mycelial growth are 25°C and 8, respectively. The optimal medium for mycelial growth is PDA medium. To our knowledge, this is the first report of D. neojuniperi causes Diplodia top dieback on Pinus thunbergii. Our results provide fundamental information for monitoring and preventing such disease in the future.