Exploring the phylogenetic diversity of Botryosphaeriaceae and Diaporthe species causing dieback and shoot blight of blueberry in Serbia.

Identifying the precise pathogens responsible for specific plant diseases is imperative for implementing targeted and efficient interventions and mitigating their spread. Dieback and shoot blight significantly diminish the lifespan and productivity of blueberries, yet the causative agents remain largely unidentified. To determine the identity and prevalence of the causal agents of branch dieback and shoot blight, we conducted multi-year and multi-site sampling of diseased highbush blueberries (Vaccinium corymbosum cv. Duke) in Serbia. Sixty-nine monosporic isolates were collected and characterized based on morphological, physiological features and multi-locus phylogenetic analysis of internal transcribed spacer (ITS), ß-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1) sequence data. Five species were identified as causal agents: Diaporthe eres (36 isolates), D. foeniculina (3 isolates), Neopestalotiopsis vaccinii (9 isolates), N. rosae (6 isolates) and Neofusicoccum parvum (15 isolates). The results of the pathogenicity tests performed with the 23 representative isolates confirmed the role of these species as primary pathogens in causing dieback and shoot blight of blueberry, with N. parvum being the most aggressive and D. eres the least. Our study underscores the diversity of genera and species of ascomycetes capable of causing blueberry dieback and shoot blight. Furthermore, our findings indicate that the agents responsible for the disease in Serbia differ from those identified in other regions worldwide.

First report of powdery mildew of blackberry caused by Podosphaera aphanis in Serbia.

Blackberries (Rubus L. subgenus Rubus Watson) are popular, wild fruits with high content of antioxidants and thus with beneficial effect on the human health (Reyes-Carmona et al. 2005). In July 2019 and May 2020, plants with typical powdery mildew symptoms were collected in the blackberry cv. 'Triple crown' orchard (of 2 ha in size) in the vicinity of Pakovrace (Moravica District, Serbia). The symptoms observed in 2019 included mild chlorotic spots on both old and young leaves accompanied by the white powdery mildew colonies on the surface of the leaves, visible on both primorcanes and floricanes. In 2020, even more intensive symptoms occurred on fruit bearing shoots which were covered with dense white fungal growth. Heavily infected leaves turned necrotic along the edges, followed by defoliation. Disease incidence was calculated by randomly counting and rating 100 plants in four replications and estimated to be over 90% while disease severity was estimated to be over 40%. Morphological characteristics were assessed using bright-field and phase-contrast microscopy (Jankovics et al. 2011) and revealed the presence of unbranched, erect conidiophores (N=50, 75 to 200 µm) with cylindrical foot-cell and up to five short cells. Conidia were unicellular, hyaline and ellipsoid-barrel-shaped (N=50, 22.5 to 35.5 × 12.5 to 15 µm) containing fibrosin bodies (in 3% KOH). All observed characteristics resembled to Podosphaera spp. (Braun and Takamatsu 2000). The presence of chasmothecia was not recorded. Further molecular identification was conducted using internal transcribed spacer (ITS) sequence analysis of two isolates, 420G-19 and 30G-20, sampled in 2019 and 2020, respectively. Total DNA was extracted directly from epiphytic mycelium on the leaves using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. PCR amplification and sequencing were performed with primers ITS1F and ITS4 (Takamatsu et al. 2010). The nucleotide sequence of the representative isolates 420G-19 (530 bp) and 30G-20 (530 bp) (Accession No. MN914995 and MT514661) shared 100% identity, while both shared 99.49 to 99.81% nt identity with 32 Podosphaera aphanis strawberry and raspberry isolates in the GenBank (the highest 99.81% with GU942455, Harvey and Xu 2010), confirming that powdery mildew of blackberry in Serbia is caused by P. aphanis. In order to fulfill Koch's postulates, 10 rooted, healthy blackberry plants cv. 'Triple crown' were dusted with conidia of isolate 30G-20 and incubated at 23°C under the high relative humidity in the glasshouse. Healthy blackberry plants incubated in the same conditions, served as negative control. The minute white fungal colonies sharing the same microscopic features with the original isolate were visible 7-8 days post inoculation on all inoculated plants. No fungal growth was observed in the negative control. Serbia is the fourth largest blackberry producer in the world (Strik et al. 2007) and the occurrence of P. aphanis causing powdery mildew as a new pathogen is of utmost importance. P. aphanis is described as strawberry and raspberry powdery mildew pathogen with a population expressing substantial genetic diversity (Harvey and Xu 2010). The molecular data on blackberry originating isolates of P. aphanis are missing. Our study showed that P. aphanis could be destructive for blackberry in Serbia, thus representing a threat for the production of this valuable crop.

Molecular Characterization of Pseudomonas syringae pv. coriandricola and Biochemical Changes Attributable to the Pathological Response on Its Hosts Carrot, Parsley, and Parsnip.

Bacterial leaf spot caused by the plant pathogenic bacterium Pseudomonas syringae pv. coriandricola (Psc) was observed on carrot, parsnip, and parsley grown on a vegetable farm in the Vojvodina Province of Serbia. Nonfluorescent bacterial colonies were isolated from diseased leaves and characterized using different molecular techniques. Repetitive element PCR fingerprinting with five oligonucleotide primers (BOX, ERIC, GTG5, REP, and SERE) and the randomly amplified polymorphic DNA-PCR with the M13 primer revealed identical fingerprint patterns for all tested strains. Multilocus sequence analysis of four housekeeping genes (gapA, gltA, gyrB, and rpoD) showed a high degree (99.8 to 100%) of homology with sequences of Psc strains deposited in the Plant-Associated Microbes Database and NCBI database. The tested strains caused bacterial leaf spot symptoms on all three host plants. Host-strain specificity was not found in cross-pathogenicity tests, but the plant response (peroxidase induction and chlorophyll bleaching) was more pronounced in carrot and parsley than in parsnip.

Characterization of Gnomoniopsis idaeicola, the Causal Agent of Canker and Wilting of Blackberry in Serbia.

Blackberry cane diseases with the symptoms of necrosis, canker, and wilting are caused by several fungi worldwide. Surveys conducted from 2013 to 2016 in Serbia revealed the occurrence of Gnomoniopsis idaeicola, the causal agent of cane canker and wilting, which was found to be distributed in almost half of the surveyed orchards, in three blackberry cultivars, and with disease incidence of up to 80%. Wide distribution and high disease incidence suggest that G. idaeicola has been present in Serbia for some time. Out of 427 samples, a total of 65 G. idaeicola isolates were obtained (isolation rate of 34.19%). Reference isolates, originating from different localities, were conventionally and molecularly identified and characterized. G. idaeicola was detected in single and mixed infections with fungi from genera Paraconiothyrium, Colletotrichum, Diaporthe, Botryosphaeria, Botrytis, Septoria, Neofusicoccum, and Discostroma, and no diagnostically specific symptoms could be related directly to the G. idaeicola infection. In orchards solely infected with G. idaeicola, blackberry plant mortality was up to 40%, and yield loses were estimated at 50%. G. idaeicola isolates included in this study demonstrated intraspecies diversity in morphological, biological, pathogenic, and molecular features, which indicates that population in Serbia may be of different origin. This is the first record of a massive outbreak of G. idaeicola infection, illustrating its capability of harmful influence on blackberry production. This study represents the initial step in studying G. idaeicola as a new blackberry pathogen in Serbia, aiming at developing efficient control measures.