The Diplodia Tip Blight Pathogen Sphaeropsis sapinea is the Most Common Fungus in Scots Pines’ Mycobiome, Irrespective of Health Status—A Case Study from Germany

The opportunistic pathogen Sphaeropsis sapinea (≡ Diplodia sapinea) is one of the most severe pathogens in Scots pine, causing the disease Diplodia tip blight on coniferous tree species. Disease symptoms become visible when trees are weakened by stress. Sphaeropsis sapinea has an endophytic mode in its lifecycle, making it difficult to detect before disease outbreaks. This study aims to record how S. sapinea accumulates in trees of different health status and, simultaneously, monitor seasonal and age-related fluctuations in the mycobiome. We compared the mycobiome of healthy and diseased Scots pines. Twigs were sampled in June and September 2018, and filamentous fungi were isolated. The mycobiome was analyzed by high-throughput sequencing (HTS) of the ITS2 region. A PERMANOVA analysis confirmed that the mycobiome community composition significantly differed between growth years (p < 0.001) and sampling time (p < 0.001) but not between healthy and diseased trees. Sphaeropsis sapinea was the most common endophyte isolated and the second most common in the HTS data. The fungus was highly abundant in symptomless (healthy) trees, presenting in its endophytic mode. Our results highlight the ability of S. sapinea to accumulate unnoticed as an endophyte in healthy trees before the disease breaks out, representing a sudden threat to Scots pines in the future, especially with increasing drought conditions experienced by pines.

Sphaeropsis sapinea and Associated Endophytes in Scots Pine: Interactions and Effect on the Host Under Variable Water Content

The ascomycete Sphaeropsis sapinea is the causal agent of the Diplodia Tip Blight disease on pines and other conifer species. This fungus has a symptomless endophytic life stage. Disease symptoms become visible when trees have been weakened by abiotic stress, usually related to warmer temperatures and drought. Currently, this disease is observed regularly in Scots pine (Pinus sylvestris) sites in parts of Europe, such as Germany, increasing dramatically in the last decade. Changes in climatic conditions will gradually increase the damage caused by this fungus, because it is favored by elevated temperature. Thus, host trees with reduced vitality due to climate change-related environmental stress are expected to be more susceptible to an outbreak of Diplodia Tip Blight disease. There is currently no established and effective method to control S. sapinea. This project aims to reveal the nature of the endophyte community of Scots pine. Utilizing the antagonistic core community of endophytes could serve as a novel tool for disease control. Results from this study provide a starting point for new solutions to improve forest health and counter S. sapinea disease outbreaks. We screened potential antagonistic endophytes against S. sapinea and infected Scots pine seedlings with the most common endophytes and S. sapinea alone and combination. The host was stressed by limiting access to water. The antagonism study revealed 13 possible fungi with the ability to inhibit the growth of S. sapinea in vitro, for example Sydowia polyspora. None of the tested co-infected fungi (Desmazierella acicola, Didymellaceae sp., Microsphaeropsis olivacea, Sydowia polyspora, and Truncatella conorum-piceae) showed strong necrosis development in vivo, even when host stress increased due to drought. However, the infection experiment demonstrated that drought conditions enhance the effect of the disease outbreak, triggering S. sapinea to cause more necrosis in the infected twigs.

Sphaeropsis sapinea found as symptomless endophyte in Finland

Sphaeropsis sapinea Diplodia sapinea Pinus sylvestris S. sapinea S. sapinea S. sapinea S. sapinea

MOLECULAR-GENETIC ANALYSIS OF PHYTOPATHOGENS IN STANDS OF THE VORONEZH REGION

This paper presents the results of DNA diagnostics of phytopathogens in the Voronezh region. DNA diagnostics was carried out step by step: isolation of total DNA from the sample by CTAB method, amplification of marker regions of phytopathogenic organisms using primers ITS1 and ITS4, electrophoretic separation of the obtained amplicons in 2% agarose gel followed by staining with ethidium bromide, determination of the nucleotide sequence of the amplified loci ABI Prism 310. The study identified the following plant diseases: Sphaeropsis sapinea, Rhizoctonia solani, Cladosporium herbarum. Along with this, we identified the Neocatenulostroma pathogen, which had not previously been found in the territories under its jurisdiction. This disease cannot be determined by phenological signs. The degree of infection by pathogens ranged from 15 to 40%. At present, the problem of protecting plants from diseases is especially urgent. It has been established that the greatest damage to forestry activities is caused by fungal and infectious diseases. At the same time, among phytopathogens, about 97% are fungal infections, 2% are bacterial and 1% are viral.

The Opportunistic Pathogen Sphaeropsis Sapinea is Found to be one of the Most Abundant Fungi in Symptomless and Diseased Scots Pine in Central-Europe

Background: The opportunistic and latent pathogen Sphaeropsis sapinea is one of the most important forest pathogens on pine. The fungus may cause Diplodia tip blight on several coniferous trees and disease symptoms come visible when trees are weakened by stress, usually related to injuries and drought. This project compares the mycobiome of healthy and diseased Scots pines. Twigs were sampled in June and September 2018 in a German forest stand with varying health status of the sampled Scots pines. Growth of 2017 and 2018 were sampled and cultivable, filamentous fungi isolated and the mycobiome was analysed by high-throughput sequencing (HTS) of the internal transcribed spacer 2 (ITS2) region. Results: A PERMANOVA analysis confirmed that the mycobiome community composition significantly differed between growth years (p < 0.001) and sampling time (p < 0.001) but not between healthy and diseased trees. Higher amount of S. sapinea was observed in June and the growth year 2017. Besides yeasts which were neglected, 23 ascomyceteous fungal endophytes were isolated. S. sapinea was the most common endophyte isolated and the second common in HTS data. It was highly abundant in symptomless (healthy) trees. Conclusion: Results highlights the ability of S. sapinea to accumulate unnoticed before disease outbreaks, implementing the sudden threat for Scots pines in the future.

Pine engraver beetle Ips acuminatus as a potential vector of Sphaeropsis sapinea

The pine engraver beetle Ips acuminatus Gyll. is a potential vector of the Sphaeropsis tip blight pathogen according to Leach’s postulates. The specimens of I. acuminatus were associated with numerous fungi species, namely Sphaeropsis sapinea (Fr.) Dyko & B. Sutton and ophiostomatoid species. The association between opportunistic pathogen S. sapinea and I. acuminatus has been confirmed for 62.9 % of all branches (44 % of needle samples and 82 % of wood samples). The presence of S. sapinea in the galleries and on the surface of the beetle indicates that I. acuminatus may transport the pathogen and later introduce it into healthy trees. The bark beetle can transfer pathogenic fungus during maturation feeding on the shoots of healthy pine crowns and into the branches during making galleries.

The most important mycoses of Austrian pine (Pinus nigra Arnold.) in the Avala area

Austrian pine is one of the species most often used for afforestation in our conditions. In the area of Avala, afforestation with Austrian and Scots pine began in 1899. The stands of Austrian pine located on the northern and northwestern foothills of the Avala Landscape of Outstanding Features are 115 years old, while the stands on the southern and southeastern side are about 65 years old. Two-year research in the area of Avala recorded the presence of 18 species of fungi on Austrian pine. Out of that number, 6 fungi were found on needles, 5 on needles and seeds, 2 on roots and stem bases, 1 on cones, 3 on trunks and branches and 1 species in vascular bundles. Of all the species, the greatest damage is caused by Sphaeropsis sapinea and species of the Armillaria genus.