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.

known to cause DNB: Dothistroma septosporum (Dorogin) M. Morelet and D. pini Hulbary (Barnes et al., 2004). Dothistroma septosporum and D. pini do not show consistent differences in the length of their conidia, so it is necessary the use of specific molecular markers for their discrimination and identification of the two pathogens (Barnes et al. 2004, Ioos et al., 2010. Dothistroma septosporum has a worldwide distribution and found in many parts of the world where pines are grown (Barnes et al., 2008, Bulman et al., 2013. D. pini was found from some countries in Europe including Ukraine and from the UsA (Drenkhan et al., 2016).
Dothistroma septosporum was first recorded in the Kiev region (smela town) of Ukraine on Pinus sylvestris L. by L. Kaznowski (Barnes et al., 2008). since 2004, DNB outbreak has been reported several times in the Tsjurupinsk (Kherson region) and Mykolaiv region on 30-year-old Crimean pine (P. nigra subsp. pallasiana or P. pallasiana, or P. nigra var. pallasiana (D. Don in Lamb) resulted in massive pine dieback in the southern Ukraine (Usichenko & Akulov 2005, Drenkhan et al., 2016. The natural range of P. nigra subsp. pallasiana covers the southern Carpathians and the Crimean Peninsula in Ukraine, as well as the Balkan Peninsula, Cyprus, the Black sea coast of Caucasus and Turkey (Barnes et al., 2008, Lazarević et al., 2017. Outbreak of DNB has spread in southern Ukraine and south-western Russia devastating more than 8000 ha of pine forests (Usichenko & Akulov, 2005).
Presently, DNB occurs throughout southern Ukraine and its severity appears to be increasing (Drenkhan et al., 2016) alongside climate change. It may act together with native or invasive pathogens and reduce the populations of Crimean pine (Tubby and Webber 2010, Adamson et al., 2018).
As DNB affects over 80 species of Pinus, as well as other conifers (Watt et al., 2005, Drenkhan et al., 2016, there is an urgent need to find effective methods for disease control and management, because DNB resulted in the rejection of planting susceptible Pinus species in some countries of Africa, Asia, Australasia, Europe and North America (Bulman et al., 2016). The main practices used to control of DNB in Ukraine are early detection of symptoms and signs of DNB, pathogen population monitoring. No chemicals applied in the Ukrainian forest due to the lack of permissible fungicides. However, copper and other fungicides were applied in the forest and landscape nurseries for pathogen spread prevention to uninfected areas, as well as an elimination of infected seedlings. According to Bulman et al. (2016), DNB may be effectively controlled using copper fungicides as it routinely applied in New Zeeland and Australia, or by planting non-susceptible species, as is the most common form of management in Europe. Only a few studies demonstrated the possibility of using biological control agents to reduce the impact of this highly damaging pathogen in forest tree nurseries (Allenzi et al., 2015).
The aim of the study was to find potential biological agents against Dothistroma needle blight. This study reports the following results: i) screening needle endophytes to search potential agents of biological control against pine needle pathogen Dothistroma pini; ii) artificial inoculation experiments to test the potential of two fungal strains to provide control of Crimean pine infection by D. pini.
Objects and methods. Fungal isolates and plant material. several endophytic fungi were recovered from needles of Crimean pine during summer sampling of the host's microbiome in Ukraine in 2012-2014. Asymptomatic, healthy needles were collected from trees growing in the Kherson region (46°31'36.2"N 32°32'01.3"E). Fifty individual needles were sampled in the canopy from each of ten trees of Crimean pine for a total of 500 needles. Needles were surface-sterilized by serial sterilization (1 min in 95 % ethanol, 5 min in 6 % sodium hypochlorite (NaOCl), and 30 s in 95 % ethanol) before plating onto 3 % potato dextrose agar (PDA). smaller sample groups were also taken from diseased pines that appeared to have lower disease severity. The hyphal tip of each morphologically different mycelium that emerged from a needle was subcultured and transferred to for later identification. Following incubation, fungal isolates recovered from each plant fragment were selected at random, purified and grouped based on phenotypic characteristics, e.g. colony morphology, colony colour, and growth rate. Isolates representing each fungal group of interest were selected for further identification by morphological traits (classic taxonomy) and/or rDNA sequencing.
Dothistroma pini was isolated from infected needles collected in 2012-2014 in southern Ukraine (Kherson region, 46°31'36.2"N 32°32'01.3"E), and was used in artificial inoculation experiments previously. Following recovery from the plant tissues, representative cultures of dominant genera were made according to morphotypes and stored at 4°C on PDA.
Selection of endophytic fungi antagonists to Dothistroma pini. Fungi were selected based on the commonness of their occurrence in the foliage of the host and their antagonistic activity. The in vitro selection of antagonists against D. pini was carried out on 8 % malt extract agar (MEA) medium a pairedgrowth assay. For this, mycelial discs (5 mm) of D. pini were inoculated on Petri dishes (100 mm) containing MEA medium and incubated at 28°C (photoperiod of 12 hours). Due to the slow growth of D. pini, after 15 days, the endophytic microorganisms were inoculated 50 mm from D. pini colony. The ability of a root endophyte to antagonize the pathogen was determined based on the inhibition level over a given period of time. This was achieved by assessing and measuring the concurrent growth of both the endophyte and the pathogen simultaneously on a shared MEA nutrient media surface (Fig. 1).
The inhibitory effect of each fungal endophyte on the pathogen is reflected in the spherical index (α/β) of the respective organisms (Rigerte et al., 2019). solitary К. В. Давиденко. Оцінювання грибів-ендофітів для біологічного контролю дотистромозу сосни кримської (Pinus nigra subsp. ... cultures of the respective endophyte and the pathogen were also plated-and observed-as controls in this experiment. The antagonism was detected also by the formation of an inhibition halo (Fig. 2). For dual-culture assay the null hypothesis was formulated as follows: the difference between the means of the spherical indices of pathogens under antagonism and the means of the spherical indices of pathogen controls zero (Rigerte et al., 2019). The alternative hypothesis was formulated as follows: the difference between the means of the spherical indices of pathogens under antagonism and the means of the spherical indices of pathogen controls is less than zero (Rigerte et al., 2019). The rationale for assuming this alternative hypothesis was that the spherical index of the pathogen under antagonism would be less than one (<1) while the controls, having grown in the absence of any biotic/abiotic pressure(s), should have a spherical index ≈1 (Rigerte et al., 2019). Thus, if the effect of specific endophyte is real, then the means would also record the same behaviour, and the subtraction of the means of controls from the pathogen replicates involved in the test would then be a negative number (Rigerte et al., 2019).
Plant material and fungal inoculation in planta. Two-year-old Crimean pine (P. nigra subsp. pallasiana) seedlings were grown from seeds in the state Forest Enterprise "Holoprystanske LG" Kherson region. Five hundred seedlings without any symptoms of DNB were replanted in the nurseries at the Forest Protection service enterprise "Kharkivlysozahist" (Kharkiv region, Ukraine) in March, 2017 for inoculation experiment.
A single plate of a mature culture of each selected fungus was used to obtain inoculum. To generate inoculum, approximately 20 ml of sterile distilled water was added to each Petri dish with mature culture and loosening the spores into suspension by passing sterile glass beads over the surface of the culture, yielding a spore density of from 3 × 10 4 to 7.3 × 10 9 cells (CFU) ml −1 (Tab. 1). Conidial suspensions were adjusted to c. 1 × 10 4 -1 × 10 9 spores ml −1 following replicate haemocytometer counts.
All suspensions were made to a volume of 200 ml with sterile distilled water and placed to the different spray bottles.
All pure cultures of eight selected fungi were made on MEA and PDA.
A solution of 200 ml of sterile distilled water was also included as a control. Concentrations of spores were not standardized across treatments since the fungi were so diverse and their interactions with the host and the pathogen could not be expected to be comparable.
In July 2017, P. nigra subsp. pallasiana seedlings were assigned to inoculation treatments with eight endophytic fungi, with 20 replicate plants per treatment. For in planta testing for D. pini antagonists, 10 days after inoculation of endophytic fungi, spores of D. pini were introduced into the seedlings. For each treatment, the foliage was sprayed with a hand-held atomiser until large droplets formed. Control plants were inoculated just with sterile distilled water.
To stimulate DNB development, free water was maintained on needle surfaces. For this, plants were sprayed twice a day with water for 7 days. These incubation conditions were modified from  and were designed to be optimum for the development of DNB.
The symptoms were evaluated from 60 to 120 days and the data were statistically analyzed by the one-way variance ANOVA method (test compared the means). After 60 days, five needles were collected randomly from each of five plants within each treatment group to determine the fungal inocula loads and percentage conidial germination. Four months after inoculation with D. pini, all needles of the previous year were collected and inspected under a microscope. DNB severity was assessed by calculating the percentage of needles with D. pini conidiomata. DNB severity was evaluated using the assessment system of schwelm et al. (2009). Molecular detection and identification. DNA was extracted from the selected symptomatic needles representing groups of different treatments. To avoid contamination on the needle surface, needle samples were washed in 96 % ethanol for 60 seconds, 2 % sodium hypochlorite for 5 minutes and rinsed in 96 % ethanol for 30 seconds. Needles were transferred to a screw cap tube together with a screw and two nuts, freeze-dried and homogenized using a fast prep shaker (Precellys 24 Bertin Technologies). DNA was extracted following a CTAB protocol. Briefly, 1 ml of CTAB was added to each sample and incubated for one hour at 65°C. samples were centrifuged and the supernatant was transferred to new tubes and cleaned with chloroform. DNA was precipitated with isopropanol, washed with 70 % ethanol and eluted in 50 ml of milliQ water. After DNA extraction, samples were cleaned using JetQuick DNA purification kit (Genomed GmbH) and the concentration was measured with a NanoDropTM (Thermo scientific).
Conventional PCR with species specific primers was used to detect D. septosporum and D. pini in the needles, using species-specific primers Dstub2-Forward (CGAACATGGACTGAGCAAAAC) and Dstub2-Reverse (GCACGGCTCTTTCAAATGAC), and DPtef-Forward (ATTTTTCGCTGCTCGTCACT) and DPtef-Reverse (CAATGTGAGATGTTCGTCGTG), respectively (Ioos et al. 2010). The PCR reaction contained 200 mM deoxyribonucleotide triphosphates, 0.2 mM of each of the two primers, 0.0265 u/ml DreamTaq polymerase with 10X DreamTaq Green Buffer (DreamTaq Green, Thermo scientific, Waltham, MA, UsA) and MgCl2 at a final concentration of 3.25 mM. The PCR conditions included an initial denaturation step at 95°C for 10 min followed by 35 amplification cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 1 min, and thermal cycling was ended by a final extension step at 72°C for 10 min. The PCR products were purified with Qiagen DNA extraction PCR M kit (Qiagen, Hilden, Germany). PCR products were size separated on 1 % agarose gels and visualized under UV light to confirm the presence of the 231 bp D. septosporum specific bands and 191 bp D. pini specific bands (Ioos et al. 2010).
Statistical analyses. statistical analysis was carried out using the software JMP®, Version 11.0.0. sAs Institute Inc., Cary, NC, 1989-2007. Data were statistically analyzed by the one-way variance method and the Tukey-Kramer multiple comparison. Test compared the means. One-way ANOVA tests were used to assess the impact of treatment on DNB severity. If ANOVA tests were significant, post-hoc Tukey tests were used to identify which treatments differed significantly. DNB severity was log-transformed before analysis. The impact of treatment on conidial germination was assessed with Kruskal-Wallis U-tests and post hoc Mann-Whitney U-tests with Holm corrections.
Results and discussion. The diversity of endophytic fungi was assessed in healthy (site 1 and 2) and symptomatic (site 3 and 4) trees of Crimean pines.
To avoid contamination and to isolate endophytic fungi only from inner needle tissues, surface disinfection was applied. The endophytic fungal community which was isolated from needles included Alternaria sp., Cladosporium sp., Colletotrichum sp., Diaporthe sp., Dothistroma sp., Fusarium sp. (including Gibberella avenacea), Gliocladium sp., Lophodermium sp., Mariannaea elegans, Sordariomycetes sp., Trichoderma sp. mostly (Tab. 2). The number of most common and fast-growing endophytic fungi that were recovered using MEA and PDA medium was not significantly different within both categories of plants (healthy and symptomatic) evaluated.

Table 2
Pooled relative abundance of selected most common fungal taxa obtained from asymptomatic and symptomatic needles collected on Crimean pine grown in south Ukraine (Kherson region)*

Continuation of table 2
A total of 244 endophytic fungi isolated from needles were randomly picked up and this population was partially characterized by rDNA (partial 18s, ITs-1, 5.8s, ITs-2 and partial 23s) sequencing. The results (see Tab. 2) showed that the most common and fast-growing endophytic fungi associated with the Crimean pine belong mainly to Ascomycetes group (Fig. 3) being the Botryosphaeriaceae, Diaporthacea, Dothideacea, and Capnodiaceae families the most frequent. The fungi Fusarium spp. and Cladosporium spp. were the dominant genera and showed the highest diversity (see Tab. 2). No correlation between fungal groups and plant categories was observed. The frequency of fungi isolation was 0.54 and 0.75 for healthy and symptomatic plants, respectively.
Partial sequences of rDNA were aligned and the relationships between endophytic isolates were evaluated by a neighbour-joining algorithm (see Fig. 3). Using this strategy, some isolates could not be identified. Cladosporium sp. was the most common genus recovered from needles. Both Sydowia and Trichoderma spp. as well as two unidentified species were also recovered at high frequency in Dothistromainfected foliage sample and visually healthy needles. Given these findings, seven commonly isolated fungi from the microbiome of Crimean pine, including Gliocladium roseum (anamorph, Clonostachys rosea) (see Tab. 1, 3) and Bionectria sp. were selected and employed as putative disease modifiers.
Clonostachys rosea is a known parasite and antagonist of other fungi (Moraga-suazo et al., 2011) and the genus Bionectria includes destructive mycoparasites, some of which are used as biocontrol agents of fungal plant pathogens (schroers, 2001). One Trichoderma sp. strain was obtained from the Environmental sciences culture collection, Natural Research Centre (Vilnius, Lithuania) showing high activity against pine pathogens in artificial inoculation experiments previously. so, a total of eight endophytic fungi were evaluated in vitro and in vivo against D. pini.
Only two species were able to inhibit the growth of the causal agent of Dothistroma needle blight of Crimean pine in vitro and two in planta (Tab. 3).
Dothistroma needle blight severity expressed as the percentage of needles with conidiomata was significantly lower on plants treated only with Trichoderma sp. 1 and Gliocladium roseum than trees treated with either species. In contrast, DNB severity on plants treated with other fungi was not significantly different from that on plants treated with D. pini. No conidiomata were observed on control plants treated with purified water.
At 120 days after D. pini inoculation, conidial density on needles from plants treated with both Trichoderma sp. 1 and Gliocladium roseum was significantly lower than on plants treated with either antagonistic fungi (Tab. 4). There was no significant difference between the other fungal species and positive treatments in conidial density or germination at either time point. Although conidial density appeared to increase between days 60 and 120, this increase was not significant (paired t-test, p > 0.05).
Overall, disease severity varied significantly by a tree (see Tab. 3), where the tree represents the combined effects of tree resistance and fungi impact. severity varied significantly from tree to tree from as little as 1.7 % to as much as 18.1 %. Modifying effects were strongly significant (P < 0.0001), as were their interactions with trees (P < 0.0001) (Tab. 5).
It has previously been shown that endophytic communities vary spatially in the plant or may be dependent on the interaction with other endophytic or pathogenic microorganisms (Allenzi et al., 2015). Moreover, plant susceptibility to DNB is often related to the stress level of the individual and stress can arise from mismatching of the planting stock's ecological traits to the planting site, root deformities, damage, and desiccation, planting at improper depths in unsuitable soils, poor nutrient and water availability, and increased exposure to pollutants, xenobiotics and contaminants (Bulman et al., 2013(Bulman et al., , 2016. so, these endophytic fungi are ubiquitous and may increase the plant resistance by improving tolerance to drought, reducing the phytopathogen settling and promoting plant growth (Allenzi et al., 2015).    Current methods for control and management include pruning and thinning of stands to reduce humidity, planting of less susceptible or completely resistant host species and the application of copperbased and other modern fungicides (Bulman et al. 2013). With the exception of a few studies with bacteria, the possibility of using biological control agents to reduce the impact of DNB in forest tree nurseries has received little attention (Allenzi et al., 2015) and this study has increased in importance in Europe for application of biological methods in forest protection.
Plant ecosystems rely heavily on their microbial communities to optimize forest health, although this association might be a good possibility to find a balance between mutualism and disease (Rabiey et al., 2019). Endophytes might cover the capacity to directly inhibit pathogens by producing antifungal compounds (Allenzi et al., 2015, Bulman et al., 2016, Rabiey et al., 2019. Most tests and experiments have carried out in laboratory conditions, but it is unknown how the endophyte-pathogen interaction will alter in the presence of changing environmental conditions and competition with other organisms in the tree system (Rabiey et al., 2019).
Thus, much more field experiment should be taken place to recognize and confirm the optimal time and conditions for usage of biocontrol agents, as climate conditions and tree physiology could alter efficacy and efficiency of biocontrol agents may vary greatly depending on climate and tree traits.
Conclusion. The use of endophytes as biocontrol agents resulted in that Dothistroma needle blight was reduced on Crimean pine seedlings treated with Trichoderma sp. and Gliocladium rosea. The significant reductions in numbers of conidia and spore germination were found on needles treated with Trichoderma sp. and Gliocladium rosea, compared to numbers following treatment with other fungi. Our result suggested that both these species may possess potential in preventing the Dothistroma needle blight at least on young pines.
Although D. pini is present almost everywhere Crimean pine is grown, both severity of disease and area of outbreak vary significantly over time and efficiency of the biocontrol agent application may vary greatly depending on climate and tree traits. That's why it is important to continue the search of endophytic biological control agents that may alter the microbial community of the host tree and could decrease DNB virulence or enable host resistance. Further work is required on the impact of the fungal species on Dothistroma infections under nursery conditions. Acknowledgements. The research was supported by the Ministry of Education and science of Ukraine within joint Ukrainian-Lithuanian project No М/93-2018 (Biological control of forest invasive pathogens to preserve biodiversity in European woodland ecosystems). The financial support is gratefully acknowledged from the sI scholarship of the swedish Institute (sI) Visby Programme). Дотистромоз зазвичай викликають два види патогенних грибів, Dothistroma septosporum та Dothistroma pini. Дотистромоз хвої є однією із найбільш важливих інвазійних хвороб сосни у багатьох країнах. Ця хвороба нещодавно виникла в Україні й була оцінена як основна загроза для сосни кримської (Pinus nigra subsp. pallasiana); для Pinus sylvestris вона є менш загрозливою.