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.

New Insight in the Occurrence of Early Blight Disease on Potato Reveals High Distribution of Alternaria solani and Alternaria protenta in Serbia.

Early blight is an economically important disease of potato worldwide. Understanding which fungal pathogens are the causal agents of early blight and their distribution on the same host is essential to finding the best strategy for the control of this disease. Previous studies have shown that Alternaria solani is the main early blight pathogen parasitizing potato. Here, we analyzed genetic and phenotypic diversity in isolates of Alternaria spp. covering all potato production areas in Serbia. We showed that the four species of Alternaria were found in areas with different distributions of the species. The occurrence of Alternaria spp. was studied by analyzing isolates from symptomatic potato leaves during multiyear sampling. In addition to Alternaria solani, we detected three more large-spored species identified as A. linariae (syn. A. tomatophila), A. protenta, and A. grandis that were involved in early blight disease on naturally infected potato leaves in Serbia. Differentiation of species was supported by phylogeny obtained from the DNA sequences of the GAPDH, calmodulin and Rpb2 genes. Our findings present a new perspective into the population structure of large-spored Alternaria species associated with early blight disease. Within the groups of large-spored Alternaria present in Serbia, evidence of A. protenta at high frequency reveals new insight into the contribution of Alternaria species in early blight disease. This work opens new perspectives for early blight management, while the distribution of different species on the same host suggests that the etiology of disease could depend on crop organization and the presence of other Alternaria hosts in close proximity to potato plants.

Genetic Diversity of Orobanche cumana Populations in Serbia.

In this study, we report genetic characterization of Orobanche cumana, the causal agent of sunflower wilting in Serbia. The genetic diversity of this parasitic plant in Serbia was not studied before. Random amplified polymorphic DNA (RAPD) markers and partial rbcL gene sequences analysis were used to characterize the O. cumana populations at the molecular level. While phylogenetic analyses of RAPD-PCR amplicons were performed using unweighted pair-group Method analyses, rbcL gene sequences were analyzed using neigbor joining method and minimum spanning tree. Molecular analyses of RAPD-PCR analysis revealed high genetic diversity of O. cumana populations which indicated high adaptive potential of this parasitic weed in Serbia. Further analyses of rbcL gene using minimum spanning tree revealed clear differences among diverse sections of Orobanche genus. Although this molecular marker lacked the resolution to display intrapopulation diversity it could be a useful tool for understanding the evolution of this parasitic plant. Our results suggested that O. cumana has great genetic potential which can lead to differentiation of more virulent races which is important for determining crop breeding strategies for their control.

Characterization of Alternaria Species Associated with Leaf Spot Disease of Armoracia rusticana in Serbia.

Leaf spot diseases caused by Alternaria species have been reported worldwide in plants in the Brassicaceae family. However, there is little information on Alternaria species causing diseases in horseradish. In the present study, 89 Alternaria spp. isolates from Armoracia rusticana, sampled from nine districts in Serbia, were characterized based on their morphology, physiology, and molecular markers. Morphological characterization and molecular analyses based on ITS, GAPDH, Alt a 1, and PM-ATP sequences identified three distinct species associated with the disease: Alternaria brassicae, A. brassicicola, and A. alternata. For all species, growth and sporulation rates at 0, 5, 10, 15, 20, 25, 30, 35, and 40°C showed a quadratic response, with A. alternata having the widest temperature optimum (20 to 30°C) while A. brassicicola had higher optimum temperatures (20 to 25°C) than A. brassicae (15 to 20°C). To gain a better understanding of the pathogenicity of these species, the influence of leaf age, host susceptibility, and ability to infect artificially wounded and nonwounded leaves were tested. The pathogenicity test identified A. brassicicola and A. brassicae as the main causal agents of horseradish leaf spot disease. Results indicated that young and intact leaves of horseradish and cabbage were less susceptible to infection and also suggested the potential for cross-infection between these two hosts. Haplotype networks showed haplotype uniformity for A. brassicae, two haplotype groups of A. brassicicola, and eight haplotype groups of A. alternata in Serbia and suggest the possible association of some haplotypes with the geographic area. This study is the first to investigate Alternaria leaf spot disease on A. rusticana in Serbia and is the first record of A. brassicicola on horseradish in this country.

In vitro biodegradation potential of airborne Aspergilli and Penicillia.

The study addresses in vitro degradation potential of airborne Aspergillus and Penicillium/Talarmyces species originating from cultural heritage conservation premises. A series of rapid, cost effective biodegradation assays were performed to assess production of extracellular pigments, acids, and enzymes. Most of the isolates have demonstrated positive growth in at least one of the preformed tests. Strongest overall degradation potential was demonstrated for Penicillium brevicompactum, P. glabrum, and Talaromyces sayulitensis while Aspergillus domesticus, A. penicillioides, A. pseudoglaucus, and A. ruber did not exhibit positive reaction in any of the employed assays. Majority of isolates exhibited proteolytic and cellulolytic activity while carbonate dissolution was observed for only five tested fungi. Highest alteration of pH value in liquid media was documented for T. sayulitensis while A. niger and P. expansum exhibited strongest acid production on CREA. Certain isolates, mostly Penicillium species, displayed production of extracellular pigments. The results imply that many of the tested fungi have significant biodegradation capacity, indicating their potential to inflict structural and esthetic alterations on cultural heritage objects.

Characterization of Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast of Mandarin in Montenegro.

Citrus blast caused by bacterium Pseudomonas syringae is a very important disease of citrus occuring in many areas of the world, but with few data about genetic structure of the pathogen involved. Considering the above fact, this study reports genetic characterization of 43 P. syringae isolates obtained from plant tissue displaying citrus blast symptoms on mandarin (Citrus reticulata) in Montenegro, using multilocus sequence analysis of gyrB, rpoD, and gap1 gene sequences. Gene sequences from a collection of 54 reference pathotype strains of P. syringae from the Plant Associated and Environmental Microbes Database (PAMDB) was used to establish a genetic relationship with our isolates obtained from mandarin. Phylogenetic analyses of gyrB, rpoD, and gap1 gene sequences showed that P. syringae pv. syringae causes citrus blast in mandarin in Montenegro, and belongs to genomospecies 1. Genetic homogeneity of isolates suggested that the Montenegrian population might be clonal which indicates a possible common source of infection. These findings may assist in further epidemiological studies of this pathogen and for determining mandarin breeding strategies for P. syringae control.