PHYTOPHTHORA CACTORUM ( LEBERT & COHN ) J . SCHRÖT AS CAUSAL AGENT OF DIEBACK OF CHESTNUT AND APPLE TREES IN MACEDONIA

From 2013–2017, 11 chestnut populations and 16 apple orchards/plantations in Macedonia were examined for health; soil, root and bark samples were collected from trees expressing symptoms regarded as Phytophthora specific. Using leaf baits of Prunus laurocerasus and selective V8 Agar (PARPNH), 19 pure Phytophthora sp. cultures were isolated and identified as P. cactorum by ITS sequencing. Sixteen isolates were from apple trees and 3 from chestnut trees. Phylogenetic analyses suggested slight distance between P. cactorum isolates originating from chestnut trees compared to those from apple orchards. Assessment of pathogenicity using chestnuts twigs showed no differences between P. cactorum isolates from the two tree host species.


INTRODUCTION
The genus Phytophthora was first reported in 1845, when Botrytis infestans, fully described in 1876 as Phytophthora infestans (Mont) De Bary was identified as the causal agent of potato blight, the main factor causing yield losses during the infamous Great Irish Famine (1844-1886).The disease was responsible for the death of approximately 1-1.5 million people and sparked massive emigration from Ireland because of the lack of food available to ordinary people [1].Soon after these events, in 1870 Peronospora cactorum (Levert and Cohn) J. Schröt was first described as the cause of rot on the cacti Cereus giganteus and Melocactus nigrotomentosus in the Czech Republic (Lebert and Cohn, 1870, cited in [2]).This fungus-like organism (FLO) was later transferred to the genus Phytophthora.
Phytophthora cactorum is a generalist plant pathogen with a worldwide distribution.It causes a variety of symptoms on many plant hosts: dampingoff of seedlings, fruit rot, leaf and stem rot, collar and crown rot, stem canker and root rot [3].Numerous plant diseases have been attributed to this oomycete, and it has been recorded on over 200 plant species, causing disease on 150 genera (e.g.including Fagus spp., Juglans regia, Malus, Castanea sativa), in 60 plant families (Tucker, 1993;Nienhous, 1960; cited in [2]).P. cactorum causes necrosis on inoculated plants of Quercus robur [4], on apple, rhododendron and strawberry, with genetically different isolates expressing different host specificity [5], and is also one of the Phytophthora spp.complex responsible for ink disease of chestnut trees [6].The only accessible relevant data on Phytophthora species detected in Macedonia is the paper published by the European and Mediterranean Plant Protection Organization (EPPO) for presence of dying off symptoms caused by P. cryptogea, dating from 1985 [7].
The morphological characteristics used for detection of Phytophthora spp., such as dimensions and shapes of zoosporangia and oogonia, may be highly variable and often overlap between species, making identification to the species level difficult [8,9].Leonian [10] stated that P. cactorum is a species easily identified by morphological characteristics, while later, isozyme analysis and mtDNA studies showed a high level of similarity between isolates originating from different geographical locations [11,12].
In the last 15-20 years there has been an increase in the number of newly described Phytophthora species [13][14][15][16], but keys available for morphological identification are not in accord with the natural division to species level sensu stricto [17].Molecular methods applied to Phytophthora species isolates, therefore, are a necessary tool for accurate identification to the species level.
In this study, we assessed chestnut populations and apple orchards in the Republic of Macedonia for symptoms of Phytophthora sp.infections.
Bark and roots from symptomatic trees, plus samples of surrounding soils were collected for isolation of Phytophthora spp.and the pathogenicity of P. cactorum strains isolated during the study was assessed.

Collection of samples.
Between 2013 and 2017, we assessed 27 sites for presence of symptoms on apple and chestnut trees (Table 1).Soil samples were collected from four sides of symptomatic trees after removal of the soil surface organic layer using methods described previously [18][19][20].The four soil samples from a single tree, each from a pit of ca 25 × 25 × 25 cm, were mixed in sterile plastic bags, and stored at room temperature (24 °C ± 4 °C) until processed.Bark samples, taken from trunk lesions and rotten tissue (mostly from the collar area), or root fragments, were collected using a knife or axe previously surface sterilized in 70 % ethanol.Isolations.The baiting method was applied to all soil and bark samples, using fully open young plant leaves of Prunus laurocerasus as bait.Soil, 250-300 g per sample, with root fragments, was placed in plastic containers and flooded with sterile distilled water, to a depth of approx. 1 cm above the soil level, and bait leaves floated on the water surface.Containers were incubated in the dark at room temperature (24 °C ± 4 °C) and leaves observed daily for discolored lesions.When observed, small fragments (10-20 mm 2 ) were cut from the lesions and placed on selective PARPNH V8 agar (200 ml V8 juice/l, pimaricin 10 mg/l, ampicillin 200 mg/l, rifampicin 10 mg/l, pentachloronitrobenzene (PCNB) 25 mg/l, nystatin 50 mg/l and hymexazol 50 mg/l) described in Jung et al. [4], and incubated at room temperature in the dark.Cultures with morphology similar to Phytophthora were sub-cultured to fresh PDA, V8 agar or malt extract agar (MEA).
Morphological identification.Morphological characteristics of isolates were recorded after two weeks of growth in the dark on PDA, V8 agar or MEA, at room temperature (24 °C ± 4 °C).To induce production of sexual and vegetative fruiting bodies, plugs (ca. 1 cm 2 ) of young cultures were placed in non-sterile soil extract solution (NSSES) [2].After 24 hours in NSSES, plugs were washed in sterile distilled water and observed under microscope [20].Morphological structures were measured, and the identification key of Erwin & Ribeiro [2] used to identify isolates based on morphology.All structures were photographed.
Growth rate.All isolates were subjected to growth-rate trials according to the protocol described in [21].Agar plugs (2 mm 2 ) were subcultured from culture margins to Petri plates containing ca. 20 ml V8 agar amended with 0.2 % Ca-CO3 with 4 replicates per sample and incubated at 24°C ± 4°C.Growth was measured in 2 perpendicular directions after 6 days of incubation.
DNA isolation and amplification.DNA was isolated from cultures grown in the dark on PDA at room temperature (24 °C ± 4 °C).Surface mycelium was gently collected with a spatula, lyophilized and ground.DNA was extracted from 50-100 mg of lyophilized tissue per sample, using the Plant-fungi DNA isolation kit (PureLink™ Plant, Total DNA Purification Kit) following the manufacturers' in-structions.Extracted DNA was subject to PCR using ITS 4 [22] and ITS 6 [23] universal Phytophthora primers, with the following amplification conditions: initial denaturation at 95 °C for 3 min.;35 cycles of denaturation (95 °C for 30 sec.), annealing (55 °C for 30 sec.), and extension (72°C for 50 sec.);and a final extension at 72 °C for 10 minutes.Amplicons were subjected to electrophoresis on 1 % agarose gel, 1 × TBE at 120 V for 90 minutes, stained with SYBR® Safe DNA gel stain and observed under UV light.All samples with visible DNA bands ranging from 800 to 1000 bp were sequenced (Macrogen, The Netherlands) utilizing both ITS 4 and ITS 6 universal Phytophthora primers.Sequences were analyzed using DNA Dynamo and compared against accessions in the online Phytophthora database (http://www.phytophthoradb.org/;[24]).Sequences were aligned using MEGA 7, and the ClustalW Multiple alignment tool, as implemented in MEGA 7 [25].Phylogenetic trees were constructed using the maximum likelihood method implemented in MEGA 7, with 1000 bootstrap replicates.In addition to sequences obtained in this research, several sequences available on http://www.phytophthoradb.org/were utilized to compare our sequences with other available Phytophthora spp.sequences.
Pathogenicity test.For the pathogenicity test, material from dormant one year old chestnut shoots taken from a single coppice was used [26].The chestnut shoots (length 10-15 cm; width 5-15 mm) were inoculated by removing a small piece of bark and insertion of agar plugs (ca 3 × 3 mm) extracted from a fresh culture of P. cactorum.Inoculation points were covered with sterile moist cotton plugs and secured with Parafilm.Two isolates were used for inoculations; one isolated from a chestnut, the other one from an apple tree.Forty replicate inoculations were made per isolate, 20 were on 5-10 mm diam.shoots, 20 on 10-15 mm diam.shoots.Inoculated shoots were placed on sterile moist filter papers in 15 cm diam.glass Petri dishes, with 10 replicate shoots per Petri dish, and incubated in dark for 7 days at room temperature (24 °C ± 4 °C; Figure 1), after which lesion lengths were measured.Ten random samples were taken for re-isolation on selective PARPNH medium to prove that the Phytophthora isolates caused the lesions.

RESULTS AND DISCUSSION
Eighty-one soil, root and/or bark samples were collected from apple trees which exhibited disease symptoms characteristic of Phytophthora infection in 16 apple orchards.In addition, 54 soil, root and/or bark samples were collected from symptomatic chestnut trees from 11 sites.Of these, fifty cultures with morphologies resembling Phytophthora spp.were obtained on selective media.Twenty-one isolates were identified as P. cactorum by culture morphology and microscopic features.Of these 19 isolates, 16 were from apple trees, and 3 from chestnut trees.All isolates were with coralloid culture morphology (Figure 2) and an average daily growth rate of 6.5 mm when incubated at room temperature (24 °C ± 4 °C) in the dark on V8 agar.Oogonia measured 29 × 27 µm on average, whereas oospores measured 21 × 21 µm on average.Antheridia were 13 × 11 µm.The mean zoosporangia dimensions were 45 × 35 µm; chlamydospores were rare but measured 22 × 21 µm on average (Figure 3).
All Phytophthora spp.isolates obtained from apple and chestnut trees in Macedonia clustered together on the same branch of the phylogenetic tree as P. cactorum, P. hedraiandra and P. pseudotsugae.While the aforementioned species are highly similar and poorly resolved between themselves, the whole branch is highly supported with a bootstrap value of 91 (Figure 4).Nevertheless, most sequences from Macedonia were highly similar, indicating a single Phytophthora sp. was responsible for the infections in both apple orchards and chestnut forests.No differences in morphologies of cultures and the dimensions of the reproductive structures were observed between the isolates of P. cactorum originating from the two different host plant species.The pathogenic-ity tests also showed no difference between the length of the lesions on the chestnut twigs induced by the isolates originating from the two different host plant species.Lesion lengths ranged from 22 mm to 59 mm on the 5-10 mm diam.shoots, and between 30 mm and 59 mm on shoots 10-15 mm in diam.(Table 2; Figure 5, 6).These results further support the conclusion that Phytophthora isolates from Macedonia had similar growth rates and pathogenicity on chestnut, and were most likely P. cactorum, or at least within this species complex.
Regarding other countries in the region, P. cactorum has been reported as pathogen on peach, almond, apple and strawberry [27,28] as well as from cherry [29,30], all in Greece.Regarding pathogenicity, isolates originating from peach and almond trees were more aggressive than apple and strawberry isolates [31].In Bulgaria, P. cactorum has been reported on American ginseng [32], and on apple and cherry [33].The pathogenicity of P. cac-torum has been assessed on young apple trees and apple fruits [33].In Serbia P. cactorum has been reported on maple [34], in the soils of young hybrid poplar stands [35], on sycamore, walnut, common hawthorn, sessile oak, Hungarian oak, common alder, European wild pear and apple [36].Having in mind these findings and the generally accepted view of P. cactorum as a generalist pathogen, we would expect that this plant pathogen is present on numerous other plant hosts in Macedonia.Further research is needed in order to gain important data on plant hosts, as well as diversity and pathogenicity of P. cactorum in the country.

Figure 1 .
Figure 1.Inoculation of chestnut twigs for pathogenicity tests

Figure 4 .
Figure 4. Phylogenetic tree constructed using MEGA7, using maximum likelihood method and Tamura-Nei substitution model.Bootstrap values were obtained after 1000 pseudereplicates.Isolates characterized in this study are in red, while the ITS sequences, publicly available at http://www.phytophthoradb.orgare in black.

Table 1 .
List of sites assessed for presence of disease symptoms characteristic for

Table 2 .
Lengths of the lesions on chestnut twigs, induced by inoculation of P. cactorum isolates originating from the 2 plant host species