Dothistroma Needle Blight in Slovenia is caused by two cryptic species: Dothistroma pini and Dothistroma septosporum

Dothistroma Needle Blight (DNB) has been known in Slovenia since 1971, but the disease symptoms have intensified in recent years. With this study, the DNB symptoms in Slovenia are for the first time connected to both phytopathogenic Dothistroma species: D. pini and D. septosporum. Based on the ITS‐rDNA comparisons, the Slovenian isolates of D. pini group with the D. pini isolates from France, Hungary and the USA and not with the D. pini isolates from Russia and Ukraine. Both mating types MAT1 and MAT2 of D. septosporum are present in Slovenia, while for D. pini, only MAT2 was found. The current widespread occurrence of D. pini in native forest stands indicates the prolonged presence of this species in Slovenia.     

Needle blight caused by Dothistroma pini in Slovakia: distribution,host range and mating types

Dothistroma needle blight (DNB) has been observed in Slovakia during the last two decades. Up until 2017, Dothistroma septosporum has only been detected and molecularly confirmed to cause DNB in Slovakia. Here, we report the detection of Dothistroma pini at six localities around Slovakia, representing different plantation types. Four pine species (Pinus sylvestris, P. nigra, P. mugo and P. jeffreyi) were confirmed as hosts of D. pini in Slovakia, of which only P. mugo has been previously reported as host in Slovakia. Three gene regions (ITS, EF1 –α, and ß-tubulin) of each of the 13 isolates were sequenced and assigned as D. pini. Based on ITS sequences, the studied isolates represent the haplotypes Dp_HAP.1, Dp_HAP.2. Both mating types were detected but at different localities. Our results suggest that in addition to D. septosporum, D. pini may contribute to DNB also in Slovakia.     

Detection of pine needle diseases caused by Dothistroma septosporum,Dothistroma pini and Lecanosticta acicola using different methodologies

Dothistroma and Lecanosticta needle blight are among the most damaging foliage diseases in plantations and natural pine stands worldwide, and are therefore listed as quarantine organisms in numerous countries. The aim of this study was to validate different methodologies used for the detection of both diseases. Based on multiplex quantitative PCR (qPCR), simultaneous detection and discrimination of the three pathogens Dothistroma septosporum, Dothistroma pini and Lecanosticta acicola were possible in symptomatic needles. Additionally, the same set of needles was analysed for the presence of all three pathogens using novel end‐point PCR assays followed by enzymatic digestion of PCR products. Results were compared with the qPCR data, and for validation, collected needles were screened morphologically for the presence of typical fruiting bodies and spores. Differences in detection sensitivity were found between the detection tools. The qPCR‐based detection allowed for specific and efficient identification and quantification of all three pathogens simultaneously. At the same time, conventional PCR assays, which target the multicopy ITS region, showed increased detection sensitivity and can be conducted without the use of expensive infrastructure and reagents.     

Dothistromin biosynthesis genes allow inter‐ and intraspecific differentiation between Dothistroma pine needle blight fungi

Dothistroma septosporum and D. pini are the causal agents of Dothistroma needle blight (DNB) of Pinus spp. in natural forests and plantations. The main aim of this study was to develop molecular diagnostic procedures to distinguish between isolates within D. septosporum, for use in biosecurity and forest health surveillance programmes. This is of particular interest for New Zealand where the population is clonal and introduction of a new isolate of the opposite mating type could have serious consequences. Areas of diversity in the dothistromin toxin gene clusters were identified in D. septosporum (51 isolates) and D. pini (6 isolates) and used as the basis of two types of diagnostic tests. PCR‐restriction fragment length polymorphism (RFLP) of part of the dothistromin polyketide synthase gene (pksA) enabled distinction between two groups of D. septosporum isolates (A and B) as well as distinguishing D. septosporum and D. pini. The intergenic region between the epoA and avfA genes allowed further resolution between some of the A group isolates in RFLP assays. These regions were analysed further to develop a rapid real‐time PCR method for diagnosis by high‐resolution melting (HRM) curve analysis. The pksA gene enabled rapid discrimination between D. septosporum and D. pini, whilst the epoA–avfA region distinguished the New Zealand isolate from most other isolates in the collection, including some isolates from DNB epidemics in Canada and Europe. Although this study is focused on differences between the New Zealand isolate and other global isolates, this type of diagnostic system could be used more generally for high‐throughput screening of D. septosporum isolates.     

The hosts and geographic range of Dothistroma needle blight in Slovakia

The occurrence and distribution of Dothistroma needle blight (DNB) were studied in 2014–2017 around Slovakia. A total of 84 localities, both native and planted, were investigated, and the presence of DNB was confirmed in 73 of them. In all positive locations, symptoms typical of DNB were observed and the Dothistroma species was confirmed using species‐specific primers either from fungal cultures or directly from needles. Both Dothistroma species—D. septosporum and D. pini—were identified. Both species occurred together in 29 locations, only D. septosporum in 42 and only D. pini in two locations. The host range of D. septosporum included 10 pine species and two spruce species. The host range of D. pini comprised the same number of pine hosts but only one spruce species. Five pine hosts, P. aristata, P. coulteri, P. densiflora, P. jeffreyi, P. × schwerinii, and one spruce host P. abies are new hosts species of D. pini. P. densiflora and Picea pungens have earlier been reported to be susceptible for DNB. In this study, D. septosporum was found from both tree species.     

The hosts and potential geographic range of Dothistroma needle blight

Dothistroma needle blight, one of the most important foliar diseases of Pinus spp., is caused primarily by the fungus Dothistroma septosporum (Dorog.) Morelet, and to a lesser extent by Dothistroma pini Hulbary. The potential distribution and abundance of Dothistroma spp. was determined by (i) developing a process-oriented model of potential distribution of Dothistroma spp. from known locations, (ii) compiling a comprehensive list of susceptible host species from existing scientific literature and (iii) determining the distribution of susceptible hosts in areas predicted to be suitable for range expansion of Dothistroma spp. Using these three sources of information regions at risk were identified as those that were predicted to be suitable for range expansion by Dothistroma spp. and included significant areas of susceptible host species.     

THE DEVELOPMENT PATTERN OF DOTHISTROMA NEEDLE BLIGHT AND ITS CONTROL

From field investigations and inoculations,Dothistroma pini,the pathogen ofDothistroma needle blight was found to overwinter as mycelium and unmatured acervuli in infectedneedles.The conidia were spreaded mainly by rain splash.The disease became widespread by trans-porting discased seedlings and seions conidia spread throughout the growing season,but mainlyfrom May to July.The diseade was found in both stand locations in each summer of investigation.The outbreak severity was serious in pure stands of susceptible pine and mixed stands with largecomponents of suseeotuble pine.but slight in mixed stands of unsusceptible species with small com-ponents of suseeptible hosts.The disease was less severe in pruned stands than in unpruned stands.The disease was most serious near the infection centre.indoor and outdoor tests,it was shown thatChlorothalonil smoke,Chlorothalonil spray and Thiram and Asomate sprays were effective controlfungicides.     

Clonal selection for resistance to Dothistroma needle blight in Pinus radiata

Fifty-two clones in twenty-two full-sib families were represented in a field trial. All cuttings were taken from hedged ortets which were four years from seed, but had been cut back periodically to one metre high since age two. One-year-old seedlings, from a seed orchard, where most of the parents of the clones were represented, were used as controls. The trial was assessed for Dothistroma needle blight infection six years after planting. The mean infection level of all the cuttings was 21.1%, compared to 29.8% for the seedlings. This difference was attributed to the greater maturation age of the cuttings. It was calculated that if the best 10% of the clones were selected, infection could have been further reduced to 12%, a level where other disease control measures would be considered unnecessary.     

The hemibiotrophic lifestyle of the fungal pine pathogen Dothistroma septosporum

Dothistroma needle blight is a forest disease of increasing international importance due to its ability to kill as well as to retard the growth of pines. It is caused by fungi in the genus Dothistroma that produce dothistromin, a non‐host selective toxin and virulence factor that is involved in necrosis of pine tissue. Recent studies of the genome of one of the main pathogenic species, Dothistroma septosporum, showed that it contains many similarities to that of the biotroph Cladosporium fulvum, including the presence of candidate biotrophic effector genes, which supported the hypothesis that D. septosporum has a hemi‐biotrophic lifestyle. Using Pinus radiata as a host, we used a combination of microscopy, histological and molecular tools to further test this hypothesis and to determine the stage of the disease cycle in which dothistromin toxin is produced. The results showed a biotrophic‐type phase in which the fungus grew over the needle surface, penetrated through stomatal pores and colonized epistomatal chambers. The subsequent necrotrophic phase was characterized by colonization of the mesophyll and production of dothistromin, with a >100‐fold increase in dothistromin levels from early necrotic lesion to sporulating lesion stages. This is consistent with the role of dothistromin as a virulence factor that is involved in lesion expansion.     

Relative susceptibility to blister rust caused by Cronartium flaccidum of several species of pine

At Florence, Italy, several species of pine were experimentally infected with blister rust. Inoculations were carried out on 3 and 15 months-old seedlings. After antificial and natural inoculation, Brutia, Aleppo, Austrian, Swiss mountain, Maritime and Italian stone pine showed pyenia and aecia. Spotted seedlings of Ponderosa pine showed only mycelium of C. flaccidum in needle and stem tissues. The exotic species seemed to have a very high degree of resistance to blister rust.     

樟子松枯梢病的防治研究

樟子松移植苗枯梢病Sphaeropsis sapinea可以通过药剂有效地进行防治。移植前以50％多菌灵wp500倍液浸苗；在人工林中，内吸性杀菌剂－甲基托布津、多菌灵为首选药剂；在树木新梢生长停止期和展叶中期（辽西北为6月初和6月中旬），70％甲基托布津wp 1000倍液或50％多菌灵wp 500倍液防治2次。保护性杀菌剂－百菌清效果不佳。适时的卫生伐对该病有明显的抑制作用。     

New host and country records of the Dothistroma needle blight pathogens from Europe and Asia

Dothistroma needle blight (DNB) is a serious disease of pines (Pinus spp.), with a worldwide distribution. It is caused by the ascomycete fungi Dothistroma septosporum (teleomorph: Mycosphaerella pini) and Dothistroma pini (teleomorph unknown). Recently, DNB was found on Pinus peuce in Austria, Pinus pallasiana in Ukraine and the European part of south-western Russia, as well as on Pinus radiata and Pinus wallichiana in Bhutan. Based on DNA sequence comparisons of the internal transcribed spacer and β-tubulin gene regions, isolates from Austria and Bhutan were identified as D. septosporum, while isolates from Ukraine and south-western Russia were identified as D. pini. Additional isolates studied from Pinus mugo in Hungary confirmed the presence of D. septosporum in this country. The record of D. septosporum on exotic P. peuce in Austria represents a new host report of this needle blight pathogen in Europe. Likewise, DNB and the associated pathogen, D. septosporum are reported from Bhutan, eastern Himalayas, for the first time. In addition, D. pini was found in two European countries and on a new host, P. pallasiana. These European records represent the only reports of D. pini from outside the north-central USA. Morphological examination of selected specimens from different hosts and countries showed that D. septosporum and D. pini overlap in the length of their conidia, while the width is slightly wider in D. pini than in D. septosporum. The differences in conidial width are so small, however, that identification of the two Dothistroma species solely based on morphology is virtually impossible. The new host and country records provided here are consistent with the continuing trend of reports of the DNB pathogens from new hosts and new geographical areas during the last two decades, particularly in the northern hemisphere.     

Variation in susceptibility of six pine species and hybrids to pitch canker caused by Fusarium circinatum

Six pine species or hybrids were tested for susceptibility to pitch canker caused by Fusarium  circinatum. Pinus  densiflora, Pinus  thunbergii, Pinus  x rigitaeda (Pinus  rigida × Pinus  taeda), P. rigida × P. x rigitaeda, Pinus  echinata and Pinus  virginiana were inoculated with three spore loads (50, 500 and 5000 per tree) of F. circinatum. External symptoms, lesion length, and the frequency of reisolation of the fungus were investigated. External symptoms were greatest in P. echinata, followed by P. virginiana, however, P. densiflora was not susceptible to F. circinatum. Based on mean lesion lengths, the six pine species or hybrids differed significantly (p < 0.01) in susceptibility to pitch canker. Pinus  echinata sustained the longest lesions, whereas P. densiflora sustained the shortest lesions. The effect of inoculum density was not significant among three spore treatments within species (p = 0.17), although lesion length was slightly greater at higher spore loads over all pine species. The fungus was reisolated from inoculated stems of all pine species tested, even on trees showing little or no damage from the disease. Additional studies are needed to further explore the basis for resistance to pitch canker.     

Needle polyamine concentrations and potassium nutrition in Scots pine

The response of free polyamines (putrescine, spermidine and spermine) in needles of Scots pine (Pinus sylvestris L.) to varying needle potassium concentrations was investigated in two potassium fertilization experiments on drained peatlands. A significant negative correlation was observed between putrescine and potassium concentrations in needles. Putrescine responded more sensitively to decreasing needle K concentrations during the growing season than during the winter. Putrescine accumulation started when needle potassium concentrations were above 5.5 mg g(dw) (-1) in summer and above 5.0 mg g(dw) (-1) in winter. A decrease in needle potassium concentrations below 4.3 mg g(dw) (-1) in summer and below 3.5 mg g(dw) (-1) in winter resulted in an exponential increase in putrescine concentrations. Putrescine accumulation was initiated well above the needle potassium concentrations generally considered indicative of deficiency. The exponential increase in putrescine concentrations in winter occurred within the range of needle potassium concentrations that has been identified as indicating severe potassium deficiency.     

Invasive brown spot needle blight caused by Lecanosticta acicola in Estonia

Lecanosticta acicola (Thüm.) Syd., a serious foliage pathogen of pines in many regions of the world, is an emerging invasive species in northern Europe. After the first record of L. acicola on Pinus ponderosa Dougl. ex Laws. in northern Estonia in 2008, monitoring was started to investigate the spread and host range of the fungus in the country. By the beginning of 2015, L. acicola was also recorded on P. uncinata Mill. ex Mirb., P. mugo Turra and on P. mugo var. pumilio (Haenke) Zenari, being the northernmost records of the fungus in Europe. So far, the single native pine species Pinus sylvestris L. has not been found to be infected. Molecular analysis proved infection of L. acicola on pines in five different localities of Estonia: in Tallinn (mainly in the Botanic Garden, northern Estonia), in Tori and Kärdla (western Estonia), in Vasula and Kärevere (east-central Estonia). The sexual state (teleomorph) of the fungus was not found, but the existence of the both mating types, MAT1-1 and MAT1-2, was confirmed.     

Shoot dieback of lodgepole pine in Scotland caused by Ramichloridium pini

Research conducted on shoot dieback of lodgepole pine, Pinus contorta, during the early 1980's in west Scotland is summarised. The dieback symptoms and their stages of development are described. Ramichloridium pini de Hoog and Rahman was regularly isolated from bark and xylem tissue of dieased shoots. Artificial inoculations established that this fungus was the cause of the disease.     

Bacterial shoot blight of sweet crab apple caused by Pseudomonas viridiflava

Sweet crab apple (Malus coronaria) is a popular ornamental tree planted in gardens and urban forests in South Korea. In 2018, severe symptoms were observed on street plantings of sweet crab apple in Jinju, South Korea including shoot blight: shoot tips, twigs and foliage of infected trees turned brown, and dried completely. The causal agent isolated from the disease lesions was identified as Pseudomonas viridiflava based on biochemical tests, pathogenicity, and 16S rRNA and rpoD gene sequencing. On artificial inoculation, this pathogen not only caused shoot blight on sweet crab apple, but also bulb rot and leaf blight on onion. Here, we report the occurrence of bacterial shoot blight caused by P. viridiflava in sweet crab apple for the first time in the world.     

Dothistroma (red‐band) needle blight of pines and the dothistromin toxin: a review

Dothistroma (red‐band) needle blight has been a problem in plantations of exotic pines in the southern hemisphere for many decades. The prevalence of this disease is currently increasing in the northern hemisphere and is now affecting trees in their native ranges. The fungal pathogen Mycosphaerella pini with its anamorph Dothistroma pini, which is responsible for the disease, produces a toxin, dothistromin, that is closely related to the potent carcinogen, aflatoxin. Understandably this has provoked concern about possible effects on the health of forestry workers. This review gives a broad coverage of literature on both disease and toxin. The fungus has a complicated taxonomy with many synonyms and in most countries only the anamorph is found. It is a necrotrophic pathogen that kills needle tissue and completes its life cycle in the lesion thus formed. Dispersal of the disease is normally by rain splash of conidiospores but there is evidence that long range dispersal has occurred by transport of contaminated plant tissue and by wind/cloud dispersal of spores in air currents. The severity of disease is affected by humidity, temperature and light. There is variation in susceptibility of different Pinus species and some achieve increased resistance with age. The current method of control in southern hemisphere plantation forests is through spraying with copper fungicides and, with P. radiata, increased disease resistance has been achieved through a breeding programme. The dothistromin toxin is a difuroanthraquinone and is similar in structure to the aflatoxin precursor versicolorin B. Part of a gene cluster encoding dothistromin biosynthetic genes has been cloned and this has confirmed parallels between the dothistromin and aflatoxin biosynthetic pathways. Dothistromin produces damaging oxygen radicals by reductive oxygen activation rather than by photosensitization, but is also thought to exert its toxic effects on specific cellular targets. Studies have shown that dothistromin is a weak mutagen and clastogen and is therefore a potential carcinogen. Although the risks to forest workers are considered very low it is prudent to avoid unnecessary exposure during periods when dothistromin levels are likely to be at their peak.     

木霉菌和毛壳菌对板栗疫病的抗生作用

哈茨木霉Trichoderma harzianumT88菌株、深绿木霉T.atrovorideT95菌株和螺旋毛壳Chaetomium spiraleND35菌株与板栗疫病菌Cryphonectria parasiticaLY菌株的平板对峙培养结果表明,木霉菌对营养和空间有强烈的竞争作用,表现在可使病原菌菌丝消解,对病菌菌丝的生长有明显的抑制作用;毛壳菌生长速度较慢,但可形成抑菌带,使病菌不扩展。室内水培枝条接种试验结果表明,接种拮抗菌的拮抗效果好,而且先接种拮抗菌后接种病原菌的处理比拮抗菌和病原菌同时接种的处理效果还好,毛壳菌没有木霉菌的作用明显。接种枝条的木霉菌、毛壳菌和病原菌的再分离结果表明,T95和ND35的定殖能力强。     

早酥梨叶枯病危害损失与防治指标研究

梨叶枯病是在早酥梨上发现的一种新病害，病原菌为Ｍｙｃｏｖｅｌｌｏｓｉｅｌｌａ ｐｙｒｉｃｏｌａ。通过调查早酥梨叶枯病在田间的消长动态，实测不同病情指数下１年生早酥梨苗的高、粗生长量、７－８年生结果树平均单果重、单株产量，建立了苗期生长量和结果树产量损失估计模型，推算出该病害防治指标病指苗期为１２．７４％，结果树为１３．８４％。