New data on the spread of the invasive species Dothistroma septosporum (Dorogin) M. Morelet in Belarus

Red band needle blight, or Dothistroma needle blight is one of the most common and harmful diseases of pine. The causative agents of the disease are pathogenic micromycetes Dothistroma septosporum (Dorogin) M. Morelet and Dothistroma pini Hulbary. Dothistroma needle blight was firstly detected in Belarus in 2012 year, but till now information about this disease in the republic is fragmentary. The article presents the results of a survey of different pine trees, carried out in the period 2016–2020 years in botanical and dendrological gardens, forest nurseries and mini-arboretums at forestry enterprises, urban stands, nurseries of decorative plants, garden centers, for the presence of Dothistroma needle blight. The species identification of the causative agent of the disease was carried out by mycological and molecular genetic methods. In this study, Dothistroma needle blight was revealed on individual trees of Pinus mugo, P. nigra and P. ponderosa in the stands of the Central Botanical Garden of the NAS of Belarus, the dendrological garden of the Glubokoe experimental forestry enterprise, in the nurseries of decorative plants in the Grodno and Minsk regions. In the collected samples of needles, the invasive species Dothistroma septosporum was identified. The frequency of occurrence of the pathogen was 4.8–7.2 %, the proportion of observation sites in which this disease was detected at 60 %. The detection of Dothistroma needle blight on pine trees, mainly on planting material imported from abroad, indicates a transboundary route of D. septosporum entering the country. Analysis of literature data indicates the potential danger of Dothistroma needle blight for pine stands in the republic, which in turn requires the organization of regular monitoring of the disease and the development of methods to limit the spread of D. septosporum in the republic.

Rapid Detection of Pine Pathogens Lecanosticta acicola, Dothistroma pini and D. septosporum on Needles by Probe-Based LAMP Assays

Needle blights are serious needle fungal diseases affecting pines both in natural and productive forests. Among needle blight agents, the ascomycetes Lecanosticta acicola, Dothistroma pini and D. septosporum are of particular concern. These pathogens need specific, fast and accurate diagnostics since they are regulated species in many countries and may require differential management measures. Due to the similarities in fungal morphology and the symptoms they elicit, these species are hard to distinguish using morphological characteristics. The symptoms can also be confused with those caused by insects or abiotic agents. DNA-based detection is therefore recommended. However, the specific PCR assays that have been produced to date for the differential diagnosis of these pathogens can be applied only in a well-furnished laboratory and the procedure takes a relatively long execution time. Surveillance and forest protection would benefit from a faster diagnostic method, such as a loop-mediated isothermal amplification (LAMP) assay, which requires less sophisticated equipment and can also be deployed directly on-site using portable devices. LAMP assays for the rapid and early detection of L. acicola, D. pini and D. septosporum were developed in this work. Species-specific LAMP primers and fluorescent assimilating probes were designed for each assay, targeting the beta tubulin (β-tub2) gene for the two Dothistroma species and the elongation factor (EF-1α) region for L. acicola. Each reaction detected its respective pathogen rapidly and with high specificity and sensitivity in DNA extracts from both pure fungal cultures and directly from infected pine needles. These qualities and the compatibility with inexpensive portable instrumentation position these LAMP assays as an effective method for routine phytosanitary control of plant material in real time, and they could profitably assist the management of L. acicola, D. pini and D. septosporum.

Development of a Rapid Loop-Mediated Isothermal Amplification Assay for the Detection of Dothistroma septosporum

A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.

Evaluation of fungal endophytes to biological control of Dothistroma needle blight on Pinus nigra subsp. pallasiana (Crimean pine)

Dothistroma needle blight (DNB), caused by Dothistroma septosporum and Dothistroma pini, is the most important forest disease of pine in many countries. This disease has recently emerged in Ukraine as a major threat to mostly Pinus nigra subsp. pallasiana and less to Scots pine. There is increasing evidence that some fungal and bacterial isolates can reduce the growth and pathogenicity of fungal plant pathogens. In this research, infected needles were collected from 30-year-old Crimean pine (P. nigra subsp. pallasiana) in four locations in Southern Ukraine. In total, 244 of endophytic fungi were recovered from needles of Crimean pine during summer sampling of the host’s microbiome in Ukraine in 2012-2014. Dothistroma spp. were detected using fungal isolation and species-specific priming PCR techniques. Among all endophytes, eight fungal species were selected based on the commonness of their occurrence in the foliage of the host and their antagonistic activity. All selected species were tested for their antifungal activity against Dothistroma needle blight. Good antifungal activity against Dothistroma pini was achieved with the Trichoderma sp. and Gliocladium rosea, indicating their good potential possibility in preventing the Dothistroma needle blight on young pines. Moreover, the significant reduction in numbers of conidia and spore germination was found on needles treated with Trichoderma sp. and Gliocladium rosea, compared to conidia numbers following treatment with other fungi. Thus, the use of an effective biological control agent against Dothistroma could be of value in forest nurseries, where it is essential to reduce losses to D. pini infection prior to transferring pines to field sites for planting out.

Four New Host and Three New State Records of Dothistroma Needle Blight Caused by Dothistroma pini in the United States

During 2010 and 2011, Dothistroma needle blight (DNB), also known as red band needle blight, was observed for the first time in Cass and Pembina counties in North Dakota (ND). In Pembina Co., DNB was observed in two sites in the Jay V. Wessels Wildlife Management Area (JWWMA). In September 2009, yellow spots on green needles were observed on some trees along the western edge of one planting. By June 2010, DNB was found on third- and fourth-year needles in both JWWMA plantings. Symptoms had developed into dark brown bands or spots on necrotic needles that contained erumpent black acervuli. In June 2011, similar DNB symptoms were observed on Pinus nigra, P. flexilis, P. ponderosa, P. cembra, and P. albicaulis in the Dale E. Herman Research Arboretum, Cass Co., ND. DNB was collected in July 2011 in Brookings Co., South Dakota (SD), from a seed source provenance planting of P. ponderosa. To identify the species causing the infections, symptomatic needles were collected in 2010 from both sites in JWWMA and then again from all four locations in 2011 on all pine species infected. Needles of P. nigra from a private residence near Fairland in Shelby County, Indiana (IN), were also included in the sample set. The rDNA-ITS was PCR-amplified either directly from conidia obtained from acervuli on the needles or from cultures obtained from isolations. Amplicons were sequenced and a BLAST search was performed in GenBank. The sequences of samples obtained from P. nigra, P. flexilis, P. cembra, and P. albicaulis in ND, P. ponderosa in SD, and P. nigra from IN showed 100% sequence homology with Dothistroma pini (Accession No. AY808302). These isolates were identical to all previously assayed isolates of D. pini from Nebraska, Minnesota, and Michigan in the United States. The P. ponderosa isolates from all three sites in ND differed from the other isolates and contained a 1-bp point mutation from a C to a T at site 72 (sequence deposited in GenBank, accession KJ933441). Mating type was determined using species-specific mating type primers for D. pini (3). All 26 samples from ND and SD were of the MAT-1 idiomorph, while the sample from IN contained the MAT-2 idiomorph. All cultures are maintained at FABI, University of Pretoria, South Africa. The two species that cause DNB, D. septosporum (G. Dorog.) M. Morelet and D. pini Hulbary, are morphologically indistinguishable and molecular characterization remains essential for correct species identification (1). Host and geographical distribution range determinations of Dothistroma spp. made without molecular methods are not valid. To date, species confirmed using DNA sequences in the United States include D. septosporum in the Pacific Northwest states of Oregon and Idaho on P. ponderosa, in Montana on P. contorta v. latifolia, and D. pini in the North Central states of Nebraska, Minnesota, and Michigan on P. nigra (1). This study documents the presence of D. pini in three additional states, including a first report of DNB in ND and SD. It also includes new records of D. pini infecting P. flexilis, P. cembra, P. albicaulis, and P. ponderosa. Results of this study have expanded the documented host range of D. pini in the United States from one (P. nigra) to five species. Globally, D. pini is now known to infect a total of 10 pine hosts (2,4,5). References: (1) I. Barnes et al. Stud. Mycol. 50:551, 2004. (2) I. Barnes et al. For. Pathol. 41:361, 2011. (3) M. Groenewald et al. Phytopathology 97:825, 2007. (4) D. Piou et al. Plant Dis. 98:841, 2014. (5) B. Piskur et al. For. Pathol. 43:518, 2013.