Diversity of Phytophthora Species Involved in New Diseases of Mountain Vegetation in Europe with the Description of Phytophthora pseudogregata sp. nov.

Abstract

New and emerging Phytophthora-related diseases in small trees, shrubs and herbaceous plants typical of subalpine vegetations have recently been observed in Italy and Slovenia. Diseased plants showed a complex symptomatology including foliar necrosis, fruit rot, shoot blight and branch bleeding cankers. Since little information is available about the aetiology of these aerial Phytophthora diseases, from 2019 to 2022, field surveys were conducted in 54 sites to define the occurrence, distribution and impact of the Phytophthora species on mountain vegetation. A total of 360 Phytophthora isolates were obtained from 397 samples collected from 33 herbaceous and woody host species. Based on phylogenetic analysis and morphometric data, 17 Phytophthora species were identified: P. pseudosyringae (201 isolates), P. plurivora (54), P. gonapodyides (21), P. ilicis (20), P. alpina (17), P. acerina (11), P. cactorum (7), P. pseudocryptogea (6), P. cambivora (5), P. idaei (4), P. psychrophila (3), P. bilorbang (2), P. chlamydospora (2), P. hedraiandra (1), P. kelmanii (1), P. rosacearum (1) and P. syringae (1). In addition, three isolates of a new putative Phytophthora species obtained from Alnus viridis, Juniperus communis and Rhododendron ferrugineum are described here as Phytophthora pseudogregata sp. nov. Overall, the results highlighted an unexpectedly high diversity of Phytophthora species in mountain areas, with many species able to cause aerial infections due to the production of caducous sporangia.

Full text

Citation: Bregant, C.; Rossetto, G.;
Meli, L.; Sasso, N.; Montecchio, L.;
Brglez, A.; Piškur, B.; Ogris, N.;
Maddau, L.; Linaldeddu, B.T.
Diversity of Phytophthora Species
Involved in New Diseases of
Mountain Vegetation in Europe with
the Description of Phytophthora
pseudogregata sp. nov.. Forests 2023,
14, 1515. https://doi.org/
10.3390/f14081515
Academic Editors: Salvatore Moricca
and Tiziana Panzavolta
Received: 29 June 2023
Revised: 23 July 2023
Accepted: 24 July 2023
Published: 25 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
Diversity of Phytophthora Species Involved in New Diseases of
Mountain Vegetation in Europe with the Description of
Phytophthora pseudogregata sp. nov.
Carlo Bregant 1, * , Giovanni Rossetto 1, Letizia Meli 1, NicolòSasso 1, Lucio Montecchio 1, Ana Brglez 2,
Barbara Piškur 2, Nikica Ogris 2, Lucia Maddau 3and Benedetto Teodoro Linaldeddu 1
1Dipartimento Territorio e Sistemi Agro-Forestali, Universitàdegli Studi di Padova, Viale dell’Università16,
35020 Legnaro, Italy; giovanni.rossetto.4@phd.unipd.it (G.R.); letizia.meli@studenti.unipd.it (L.M.);
nicolo.sasso@phd.unipd.it (N.S.); montecchio@unipd.it (L.M.); benedetto.linaldeddu@unipd.it (B.T.L.)
2Department of Forest Protection, Slovenian Forestry Institute, Veˇcna pot 2, 1000 Ljubljana, Slovenia;
ana.brglez@gozdis.si (A.B.); barbara.piskur@gozdis.si (B.P.); nikica.ogris@gozdis.si (N.O.)
3Dipartimento di Agraria, Universitàdegli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy;
lmaddau@uniss.it
*Correspondence: carlo.bregant@phd.unipd.it
Abstract:
New and emerging Phytophthora-related diseases in small trees, shrubs and herbaceous plants
typical of subalpine vegetations have recently been observed in Italy and Slovenia. Diseased plants
showed a complex symptomatology including foliar necrosis, fruit rot, shoot blight and branch bleeding
cankers. Since little information is available about the aetiology of these aerial Phytophthora diseases,
from 2019 to 2022, field surveys were conducted in 54 sites to define the occurrence, distribution
and impact of the Phytophthora species on mountain vegetation. A total of 360 Phytophthora isolates
were obtained from 397 samples collected from 33 herbaceous and woody host species. Based on
phylogenetic analysis and morphometric data, 17 Phytophthora species were identified: P. pseudosyringae
(201 isolates), P. plurivora (54), P. gonapodyides (21), P. ilicis (20), P. alpina (17), P. acerina (11), P. cactorum (7),
P. pseudocryptogea (6), P. cambivora (5), P. idaei (4), P. psychrophila (3), P. bilorbang (2), P. chlamydospora (2),
P. hedraiandra (1), P. kelmanii (1), P. rosacearum (1) and P. syringae (1). In addition, three isolates of a
new putative Phytophthora species obtained from Alnus viridis,Juniperus communis and Rhododendron
ferrugineum are described here as Phytophthora pseudogregata sp. nov. Overall, the results highlighted an
unexpectedly high diversity of Phytophthora species in mountain areas, with many species able to cause
aerial infections due to the production of caducous sporangia.
Keywords: alpine regions; emerging disease; oomycetes; foliar necrosis; ITS clade 6; phylogeny
1. Introduction
The large genus Phytophthora de Bary includes several invasive plant pathogens that
represent an increasing threat to forest ecosystems and agriculture productions world-
wide [
1
–
4
]. Over the last 20 years, scientific interest in this group of oomycetes has increased
rapidly in forest pathology and this has led to the discovery of several new species and
pathosystems [5–8].
Most of the known Phytophthora species have a soilborne or waterborne lifestyle, due
to the production of persistent sporangia and the release of motile zoospores [
9
,
10
]. The
majority of Phytophthora species are necrotrophic or hemibiotrophic pathogens, able to cause
root rot diseases in herbaceous and woody plant hosts; whereas a few species, especially
those strongly associated with water habitats, can also survive as saprophytes [
11
]. The
main symptoms caused by pathogenic Phytophthora species with a soilborne lifestyle include
fine root losses, root rot, collar necrosis and stem bleeding cankers. Plants with root
and collar infections show nonspecific secondary symptoms at the canopy level, such as
epicormic shoots and sudden death [1,12].
Forests 2023,14, 1515. https://doi.org/10.3390/f14081515 https://www.mdpi.com/journal/forests

Forests 2023,14, 1515 2 of 24
Conversely, Phytophthora species with an airborne or mixed airborne and soilborne
lifestyle have the ability to produce caducous sporangia and infect fruits, leaves, shoots,
twigs and branches, causing necrosis, rots and an anticipated loss of organs [
1
,
13
–
15
].
Caducous sporangia can act directly as infective propagules or release motile zoospores [
1
].
Aerial Phytophthora infection can occur actively via lenticels or stomata in the epigeal organs
of the host [
16
]. The ability to produce caducous sporangia is a feature common in the
species belonging to clades 1, 3, 4 and 8 [
17
]. Within this last clade, one of the most aggres-
sive species is Phytophthora ramorum, known to cause leaf blight, shoot blight and bleeding
cankers on forest and ornamental plant species in the temperate areas of North America
and Europe [
5
,
18
,
19
]. Other species belonging to clade 8, such as P. foliorum and P. hibernalis
have been reported as airborne pathogens on Rhododendron and
Citrus spp. [20–22]
. In
agriculture and horticulture, species of clades 1 and 4, such as P. cactorum,P. infestans,
P. nicotianae and P. palmivora, are well known to cause leaf, stem and fruit diseases on many
herbaceous and wood crops [23–29].
Clade 3 includes a few cryptic species characterized by a partial aerial lifestyle with
a relatively low optimum temperature for growth and a common association with native
forest species [
14
,
30
,
31
]. In particular, Phytophthora pseudosyringae is emerging as an invasive
pathogen on a broad number of hosts at global scale [14,32–34].
In Europe, aerial Phytophthora diseases have been studied mainly on agricultural
crops [
1
,
26
,
35
–
38
] and to a much lesser extent on forest trees, especially in subalpine
ecosystems [
14
]. Alpine and subalpine regions are important biodiversity hotspots for
the flora, including a large number of plants and many endemisms in very confined
environments and extreme conditions [
39
]. Due to the huge floristic diversity in small
spatial scales, mountain forests could represent useful models to understand the ecological
and evolutionary host–pathogen dynamics and to conserve pristine ecosystems [40,41].
Therefore, given the growing expansion of Phytophthora diseases in subalpine ecosys-
tems in Italy and Slovenia and the still limited information available about these pathosys-
tems, a study was conducted to isolate, identify and characterize the main pathogens
associated with these new and emerging diseases.
2. Materials and Methods
2.1. Field Survey and Sampling Procedure
Field surveys were conducted from autumn 2019 to summer 2022 in 54 sites distributed
in different mountainous areas of Northeast Italy, Sardinia and Western Slovenia (Table 1).
The monitored sites are located at an altitude ranging from the valley bottom (700 m a.s.l.)
to above the tree line (2100 m a.s.l.) and include forests, riparian ecosystems and heathlands
typical of alpine and subalpine formations.
At each site, plants were visually checked for the occurrence of typical Phytophthora
symptoms, such as leaf and fruit necrosis, shoot blight, wilting twigs, branches dieback and
bleeding cankers. In the most impacted formations, the disease incidence and mortality
rate were estimated along 25 m long linear plots. Disease incidence was calculated as
the number of symptomatic trees out of the total number of trees (DI = n/N
×
100) and
mortality as the number of dead trees out of the total number of trees (M = d/N
×
100) [
42
].
In each site, a variable number of tissue samples of leaves, twigs and branches was
collected from symptomatic plants (Table 1). Overall, 397 samples were collected from
33 host species, small trees, shrubs and herbaceous plants. These included Acer monspessu-
lanum,Acer pseudoplatanus,Alnus cordata,Alnus glutinosa,Alnus incana,Alnus viridis,Betula
pubescens,Calluna vulgaris, Erica carnea, Fagus sylvatica,Fragaria vesca,Fraxinus excelsior,
Genista corsica,Ilex aquifolium,Juniperus communis,Laburnum alpinum,Larix decidua,Lonicera
alpigena,Lycopodium clavatum,Pinus mugo,Populus tremula,Quercus pubescens,Rhododendron
ferrugineum,Rhododendron hirsutum,Rubus idaeus,Salix alpina, Salix atrocinerea,Salix caprea,
Sorbus aria,Sorbus aucuparia, Taxus baccata, Vaccinium myrtillus and Vaccinium vitis-idaea
(Table 1). The samples were sealed in plastic bags, labelled and used for Phytophthora
isolations within 24–48 h.

Forests 2023,14, 1515 3 of 24
Table 1. Study sites information, plant species monitored and number of samples collected.
Survey
Sites Country Elevation
(m a.s.l.) Geographic Coordinates Sampled Species *
1Italy 1030 46.4711671 12.4611700 Sau(3), Vm(5), Pt(4), La(2), Fe(2), Fv(2)
2Italy 912 46.4622684 12.4746272 Ai(2), Sc(1)
3Italy 1220 46.4675090 12.4833650 Fe(2), Lc(12), Fv(1)
4Italy 1060 46.4729600 12.4668290 Vm(11), Pt(6), Fe(2), Sc(1)
5Italy 1012 46.4798320 12.5178940 Fe(2), Vm(2)
6Italy 1900 46.4498739 12.5011762 Rf(2)
7Italy 1692 46.4491930 12.5041780 Pm(2), Vm(1)
8Italy 1757 46.4777890 12.5932750 Av(18), Ri(4), Pm(1)
9Italy 1691 46.4760760 12.6366770 Av(3)
10 Italy 1251 46.4926832 12.5620431 Ai(9)
11 Italy 1841 46.4852460 12.5585580 Av(2)
12 Italy 1912 46.4789100 12.5493510 Ld(1), Sau(1)
13 Italy 1752 46.5811670 12.2562680 Vm(6), Jca(3), Rf(1), Vv(1), La(1)
14 Italy 1866 46.5962680 12.2694770 Jc(2), Vm(2), Pm(1)
15 Italy 1947 46.5109880 12.3933840 Pm(5), Vm(2), Jca(1)
16 Italy 1566 46.4052340 12.4649810 Vm(3), Bp(2), Sa(1), Pm(1)
17 Italy 1725 46.6486230 12.4474240 Av(2), Sc(1)
18 Italy 1860 46.6663880 12.4912310 Vm(8), Rf(3)
19 Italy 1882 46.6644254 12.4493550 Vm(11), Vv(2), Jc(2), Cv(2), Rf(2), Pm(1), Ec(2)
20 Italy 1920 46.6008270 12.5590990 Vm(12), Jc(7), Cv(2), Rf(6), Rh(2)
21 Italy 1603 46.6025622 12.5915985 Jc(1)
22 Italy 1320 46.5898670 12.5807640 Pt(10)
23 Italy 1796 46.4067140 12.0764750 Sc(1), Av(4), Pm(2), Jc(3)
24 Italy 1074 46.0994836 46.0994836 Fe(2)
25 Italy 1550 45.9477727 12.0087351 Jc(3)
26 Italy 1670 45.9746300 11.4080100 Jc(10), Sc(2)
27 Italy 1273 45.9428700 11.4237400 Sc(6)
28 Italy 1199 45.9412300 11.4330700 Sc(2)
29 Italy 1009 45.8648319 11.5232058 Fe(1)
30 Italy 1337 45.8462570 11.7940760 Jc(4), Sar(2)
31 Italy 1760 46.3797890 13.4884770 Rf(2), Sc(2), Sa(1)
32 Italy 1355 46.3787840 13.4757220 Sa(2), Pm(1)
33 Italy 888 46.5059780 13.2630950 Ai(1)
34 Italy 1633 46.2133271 13.5278655 Sau(2), Sc(1)
35 Italy 1830 46.5753410 13.1770300 Vm(5), Rf(4), Av(2), Vv(1)
36 Italy 1990 46.5705310 13.0514810 Av(2)
37 Italy 1010 46.5379836 13.0856086 Fe(1)
38 Italy 1735 46.1515890 11.5346470 Vm(13), Rf(6)
39 Italy 1750 46.1376260 11.5419110 Vm(6), Av(1), Pt(3)
40 Italy 2065 46.8869230 12.2004166 Vm(6), Rf(4), Jc(1), Av(1)
41 Italy 1502 46.6554920 12.3509230 Vv(2), Rf(1), Pm(1)
42 Italy (Sardinia) 1125 40.0437071 9.2064231 Ia(2), Ag(3), Am(2), Qp(4)
43 Italy (Sardinia) 1328 40.0326060 9.2456210 Ia(1), Sat(1)
44 Italy (Sardinia) 1517 40.0177590 9.2788890 Jc(5), Gc(2), Ag(1)
45 Italy (Sardinia) 860 39.9386700 9.4810890 Ac(3)
46 Italy (Sardinia) 980 40.3495542 8.8807715 Ia(14)
47 Italy (Sardinia) 1029 40.4227424 8.9957414 Ia(6), Tb(1)
48 Italy (Sardinia) 825 39.9213336 9.4757911 Ag(2)
49 Slovenia 1755 46.3549947 13.9057009 Av(2), Pm(1), Ld(1), Vm(1), Sc(1), Ap(1), Sau(1)
50 Slovenia 1615 46.2378426 13.9943075 Av(3), Sar(1), Rf(1), Sc(2), Vm(1), Fs(1)
51 Slovenia 704 46.2732634 13.9879130 Ai(2)
52 Slovenia 1450 45.9788312 13.8629901 Fs(1)
53 Slovenia 1495 45.9785910 13.8643530 Pm(2)
54 Slovenia 907 45.9378821 13.9793240 Fs(1), Ap(1)
* In brackets the number of samples collected from each plant species: Acer monspessulanum (Am), Acer pseudo-
platanus (Ap), Alnus cordata (Ac), Alnus glutinosa (Ag), Alnus incana (Ai), Alnus viridis (Av), Betula pubescens (Bp),
Calluna vulgaris (Cv), Erica carnea (Ec), Fagus sylvatica (Fs), Fragaria vesca (Fv), Fraxinus excelsior (Fe), Genista corsica
(Gc), Ilex aquifolium (Ia), Juniperus communis (Jc), Laburnum alpinum (La), Larix decidua (Ld), Lonicera alpigena (La),
Lycopodium clavatum (Lc), Pinus mugo (Pm), Populus tremula (Pt), Quercus pubescens (Qp), Rhododendron ferrugineum
(Rf), Rhododendron hirsutum (Rh), Rubus idaeus (Ri), Salix alpina (Sa), Salix atrocinerea (Sat), Salix caprea (Sc), Sorbus
aria (Sar), Sorbus aucuparia (Sau), Taxus baccata (Tb), Vaccinium myrtillus (Vm) and Vaccinium vitis-idaea (Vv).

Forests 2023,14, 1515 4 of 24
2.2. Phytophthora Isolation and Characterization
Phytophthora isolation was performed directly from the symptomatic tissue samples.
Necrotic leaves were externally disinfected and cut in small pieces along the border of
active lesions, whereas shoots and bark samples from bleeding cankers, after removing
the outer bark, were cut in small fragments (along the margin of each lesion) with a sterile
scalpel. In both cases, small pieces of 3–5 mm
2
were placed on 90 mm diameter Petri
dishes containing the selective medium PDA+ [14]. In samples that resulted negative, the
procedure was repeated up to three times. After incubation at 20
◦
C for 3 days in the dark,
hyphal tips of emerging colonies were taken and transferred into new PDA and carrot agar
(CA) Petri dishes and incubated at 20 ◦C in the dark.
Isolates were morphologically examined and then grouped into morphotypes based
on colony appearance and morpho-biometric data of sporangia, oogonia, chlamydospores
and hyphal swellings. To enhance the production of sporangia, CA plugs of each isolate
were placed in Petri dishes containing pond water and asymptomatic alder roots. Petri
dishes were kept at 20
◦
C in the dark and sporangia production was assessed every 12 h
for 3 days.
For the new putative species, colony morphology was determined on 7-day-old cul-
tures incubated at 20
◦
C in the dark as reported in Bregant et al. [
14
]. Cardinal temperatures
for growth were evaluated on CA plates incubated at 2, 5, 10, 15,18, 20, 23, 25, 27, 30, 32 and
34
◦
C (
±
0.5
◦
C) in the dark. Five replicates for each isolate were made and colony diameter
was measured after 7 days. Morphology of sporangia (n. 50) and the ability to produce
hyphal swellings and chlamydospores was recorded for each isolate. Breeding system
was examined after 20 days on CA at 20
◦
C in the dark. Measurements and photos of
the morphological structures (sporangia, chlamydospores, hyphal swellings, oogonia and
anteridia) were recorded using the software Motic Images Plus 3.0 paired with a Moticam
10+ camera connected to a Motic BA410E microscope. The sizes are presented as mean
values ±standard deviation.
Representative isolates of each species were stored on PDA and CA slants under oil in
the culture collection of the Dipartimento Territorio e Sistemi Agro-Forestali, Università
degli Studi di Padova.
The ex-type culture of the new species was deposited at the Westerdijk Fungal
Biodiversity Institute, Utrecht, The Netherlands, and nomenclatural data in MycoBank
(www.MycoBank.org, accessed on 29 June 2023). The holotype was lodged with the herbar-
ium of Westerdijk Fungal Biodiversity Institute as a dried culture on CA.
2.3. Molecular Identification of the Isolates
Isolation of genomic DNA was performed from the mycelium of 7-day-old Phytoph-
thora colonies as reported in Linaldeddu et al. [
28
]. For all isolates, the internal transcribed
spacer (ITS) region of the rDNA, including the 5.8S rRNA gene, was amplified and se-
quenced using the universal primers ITS1 and ITS4 [
43
]. ITS sequences were used to
confirm the identification at species level. For three isolates of the new putative species
another two DNA regions, namely ß-tubulin (Btub) and cytochrome c oxidase subunit
I (cox1), were amplified and sequenced using the primer-pairs TUBUF2/TUBUR1 and
FM84/FM83 [
44
,
45
], respectively. Polymerase chain reactions (PCR) were performed in
50
µ
l reaction mixtures using the GoTaq
®
Hot Start Green Master Mix (Promega, Milano,
Italy) and a SimpliAmp Thermal Cycler (Thermo Fisher Scientific Inc, Waltham, MA, USA).
Amplification conditions for the three DNA regions were as follows: an initial denaturation
step at 94
◦
C for 1 min, followed by 35 cycles of denaturation at 94
◦
C for 1 min, annealing
at 55
◦
C for 1 min, extension at 72
◦
C for 1 min and a final elongation step of 7 min at 72
◦
C
for ITS; an initial denaturation step at 94
◦
C for 2 min, followed by 35 cycles of denaturation
at 9
◦
C for 40 s, annealing at 54
◦
C for 1 min, extension at 72
◦
C for 1 min and a final
elongation step of 7 min at 72
◦
C for Btub; and an initial denaturation at 95
◦
C for 2 min
followed by 38 cycles at 95
◦
C for 25 s, 53
◦
C for 50 s, 72
◦
C for 70 s and a final extension
step of 9 min at 72 ◦C for cox1.

Forests 2023,14, 1515 5 of 24
The PCR products were purified using the Monarch
TM
PCR & DNA Cleanup Kit ac-
cording to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA) and
sequenced by BMR Genomics DNA sequencing service (www.bmr-genomics.it accessed
on 23 June 2023). Sequences were edited with FinchTV v1.4.0 (Geospiza, Inc., Seattle, WA,
USA, http://www.geospiza.com/finchtv, accessed on 29 June 2023) and compared with
sequences of ex-type culture deposited in GenBank (http://blast.ncbi.nlm.nih.gov accessed
on 23 June 2023). New sequences were deposited in GenBank (Table 2).
Table 2.
Details of Phytophthora isolates included in the phylogenetic analyses. Ex-type cultures are
given in bold typeface and newly generated sequences are indicated in italics.
Species Collection No. Host GenBank Accession Number
ITS Btub Cox1
Phytophthora acerina CBS 133931 Acer pseudoplatanus JX951285 - -
P. acerina CB222 Juniperus communis OR167204 - -
P. agathidicida P15175 Agathis australis KP295308 - -
P. alpina CBS 146801 Alnus viridis MT707332 - -
P. alpina CB387 Vaccinium myrtillus OR167205 - -
P. alticola TBF0060A10 Eucalyptus grandis KX247599 - -
P. amnicola CBS 131652 water JQ029956 JQ029952 MH477740
P. amnicola VHS 19503 water JQ029958 JQ029954 JQ029950
P. asparagi VHS 17644 Lomandra sonderi EU301168 JN547592 HQ012845
P. austrocedrae CBS 122.911 Austrocedrus chilensis DQ995184 - -
P. bilorbang CBS 161653 Rubus anglicandicans JQ256377 JQ256374 MH477742
P. bilorbang SA146 R. anglicandicans JN547624 JN547585 JN547646
P. bilorbang CB600 J. communis OR167206 - -
P. borealis CBS 132023 water HM004232 JQ626615 MH136854
P. borealis AKWA 57.2-0708 water JQ626598 JQ626614 JQ626624
P. cactorum CBS 231.30 Syringa vulgaris MG783385 - -
P. cactorum CB389 Sorbus aria OR167207 - -
P. cambivora CBS 114087 Castanea sativa MG783387 MH493913 MH136860
P. cambivora CB400 Alnus incana OR167208 - -
P. captiosa CBS 119107 Eucalyptus sp. DQ297402
P. castaneae ICMP 19434 Castanea crenata KP295319 - -
P. castanetorum CBS 142299 C. sativa MF036182 - -
P. chlamydospora P6133 Prunus sp. MG865471 MH493919 MH136867
P. chlamydospora VHS 3753 soil EU301160 JN547616 HQ012878
P. chlamydospora CB480 Salix alpina OR167209 - -
P. cinnamomi CBS 144.22 Cinnamomum burmannii MG865473 - -
P. citrophthora CBS 950.87 Citrus sp. MG865476 - -
P. clandestina CBS 347.86 Trifolium subterraneum MG865477 - -
P. cocois P19948 Cocos nucifera KP295304 - -
P. crassamura PH138 Juniperus phoenicea KP863493 KX251202 KP863485
P. crassamura CB267 Cynara cardunculus MZ569853 OQ067252 OQ067256
P. dauci CBS 127102 Daucus carota KC478761 - -
P. elongata CBS 125799 Eucalyptus marginata GQ847754 - -
P. fluvialis CBS 129424 water MG865491 JN547595 MH136887
P. fluvialis VHS 17350 water EU593261 JN547593 JF701440
P. fragariaefolia CBS 135747 Fragaria ×ananassa AB819580 - -
P. gibbosa CBS 127951 Acacia pycnantha MG865499 MH493942 MH136894
P. gibbosa VHS 22008 Grevillea sp. HQ012936 JN547597 HQ012849
P. gonapodyides P7050 Alnus sp. MG865501 MH493944 MH136896
P. gonapodyides SLPA72 Eucalyptus obliqua HQ012937 JN547598 HQ012850
P. gonapodyides CB367 J. communis OR167210 - -
P. gregata CBS 127952 Patersonia sp. MG865503 MH493945 MH477746
P. gregata MJSP235 Pinus radiata EU301172 JN547602 HQ012853
P. hedraiandra CBS 111725 Viburnum sp. MG865504 - -
P. hedraiandra CB415 A. viridis OR167211 - -
P. hydrogena P19968 water KC249959 - -

Forests 2023,14, 1515 6 of 24
Table 2. Cont.
Species Collection No. Host GenBank Accession Number
ITS Btub Cox1
P. idaei CBS 971.95 Rubus idaeus MG865509 - -
P. idaei CB101 R. idaeus OR167212 - -
P. ilicis P3939 Ilex aquifolium MG865511 - -
P. ilicis CB265 I. aquifolium OR167213 - -
P. inundata CBS 216.85 Salix matsudana MG865516 MH493958 MH136910
P. ipomoeae CBS 109229 Ipomoea longipedunculata KF777191 - -
P. irrigata P16861 water MG865520
P. kelmanii CBS 146551 Ptilotus pyramidatus MT210487 - -
P. kelmanii CB426 A. incana OR167214
P. lacustris P245 S. matsundana JQ626605 JQ626619 MH136916
P. lacustris HSA1959 water HQ012956 JN547618 HQ012880
P. lilii CBS 135746 Lilium longiflorum MG865523 - -
P. litoralis CBS 127953 Banksia sp. MG865526 MH493967 MH136921
P. litoralis VHS 19173 Banksia sp. EU869199 JN547610 HQ012865
P. marrasii CBS 148033 Cynara cardunculus MZ569854 - -
P. megakarya CBS 238.83 Theobroma cacao HQ261610 - -
P. megasperma CBS 402.72 n/d MG865535 MH493973 MH136930
P. megasperma ME16 Punica granatum OP999676 OQ067253 OQ067257
P. mississippiae P19994 water MG865542 MH493980 MH136935
P. mississippiae 57J4 water KX251313 KF112853 KF112861
P. moyootj CBS 138659 soil KJ372256 KJ372303 MH477750
P. moyootj DH103 water KJ372255 KJ372301 KJ396700
P. niederhauserii P10616 Hedera helix AY550915 - -
P. ornamentata CBS 140647 Pistacia lentiscus MG865556 MN207275 MH136947
P. ornamentata PH153 P. lentiscus KP863497 MN207276 KP863487
P. palmivora CBS 305.62 Areca catechu MG865559 - -
P. pinifolia CBS 122924 Pinus radiata MG865566 MH493999 MH136958
P. pinifolia CMW 26669 P. radiata EU725807 JN935979 JN935961
P. plurivora CBS 124093 Fagus sylvatica MG865568 - -
P. plurivora CB358 J. communis OR167215 - -
P. polonica P131445 A. glutinosa DQ396410 - -
P. pseudocryptogea CBS 139749 Isopogon buxifolius KP288376 - -
P. pseudocryptogea CB482 J. communis OR167216 - -
P. pseudogregata CBS 149859 J. communis OR167217 OR189513 OR189516
P. pseudogregata CB308
Rhododendron ferrugineum
OR167218 OR189514 OR189517
P. pseudogregata CB366 Alnus viridis OR167219 OR189515 OR189518
P. pseudosyringae CBS 111772 Quercus robur MG865574 - -
P. pseudosyringae CB303 J. communis OR167220 - -
P. psychrophila CBS 803.95 Q. robur MG865576 - -
P. psychrophila CB195 Quercus pubescens OR167221 - -
P. quercina CBS 784.95 Quercus sp. MG865578 - -
P. quininea CBS 407.48 Cinchonae officinalis MG865580 - -
P. ramorum CBS 101553 Rhododendron sp. MG865581 - -
P. richardiae IMI 340618 Zantedeschia aethiopica MK496521 - -
P. riparia CBS 132024 water MG865583 JQ626607 MH136975
P. riparia VI 3-100B9F water HM004225 JQ626618 MH136975
P. rosacearum CBS 124696 Malus sp. EU925376 - -
P. rosacearum CB481 J. communis OR167222 - -
P. siskiyouensis CBS 122206 Lithocarpus densiflorus EF523386 - -
P. syringae CBS 110161 Syringa vulgaris AY230190 - -
P. syringae CB64 Salix atrocinerea OR167223 - -
P. thermophila CBS 127954 E. marginata MG865593 MH494019 MH136985
P. thermophila VHS 16164 Banksia grandis EU301158 JN547614 HQ012875
P. tyrrhenica CBS 142301 Quercus sp. KU899188 - -
P. versiformis TP13.46 Corymbia calophylla KX011279 - -
Phytophthora sp. CBS 147721 A. incana OP999674 OQ067250 OQ067254
Phytophthora sp. CB61 A. incana OP999675 OQ067251 OQ067255

Forests 2023,14, 1515 7 of 24
2.4. Phylogenetic Analysis
Molecular phylogeny based on ITS sequences was used to reconstruct evolutionary
relationships among the Phytophthora species obtained in this study into the known clades
of the genus [
17
]. Twenty ITS sequences representative of the 18 species obtained were
compiled in a dataset together with 51 sequences from ex-type material of Phytophthora
species representative of all phylogenetical clades (Table 2).
In addition, a multigene phylogeny based on concatenated ITS, Btub and cox1 se-
quences of three isolates obtained in this study and other 19 formally described Phytophthora
species in the sub-clade 6b, including ex-type cultures was performed (Table 2).
Sequences were aligned with ClustalX v. 1.83 [
46
], using the parameters reported by
Bregant et al. [14].
Phylogenetic reconstructions were performed with MEGA-X 10.1.8, including all gaps
in the analyses. The best model of DNA sequence evolution was determined automatically
by the software [
47
]. Maximum likelihood (ML) analysis was performed with a neighbour-
joining (NJ) starting tree generated by the software. A bootstrap analysis (1000 replicates)
was used to estimate the robustness of nodes. Alignments and trees are available in
TreeBase [studies S30526 and S30527].
2.5. Pathogenicity Test
The pathogenicity of the new Phytophthora species and other seven species isolated for
the first time from common juniper was tested on 5-year-old Juniperus communis seedlings
grown in plastic pots (20 cm diameter, 5 L volume). Ten 2-year branches were inoculated
with each isolate, and ten were used as control. Inoculated point was surface-disinfected
with 70% ethanol and a small piece of outer and inner bark (3
×
3 mm) was removed with
a flamed scalpel. An agar mycelium plug of the same size (3
×
3 mm) taken from the
margin of an actively growing colony (4-day-old) on PDA was placed into the wound and
the inoculation point was covered with moistened cotton and wrapped in aluminium foil.
Control seedlings were inoculated with a sterile PDA plug applied as described above.
All inoculated seedlings were kept in a cold greenhouse at 17 to 26
◦
C and watered
regularly for 30 days. At the end of the experimental period, seedlings were checked for the
presence of external and internal disease symptoms. The length of necrotic lesion surrounding
each inoculation point was measured after removing the outer bark with a scalpel.
Re-isolation was performed by transferring 10 pieces of inner bark fragments taken
around the margin of the necrotic lesions onto PDA+. Growing colonies were subcultured
onto CA, incubated in the dark at 20
◦
C for seven days and identified by morphological
and molecular analysis (ITS region).
2.6. Data Analysis
The variation in Phytophthora community structure among trees, shrubs and herba-
ceous plant species was assessed using the Jaccard similarity index (Jc) based on presence
or absence of species among different microbial communities [
48
], Jc = j/(a + b + j), where
j = represents the number of species in common between the two groups; a = the number
of species isolated from group A; b = number of species isolated from group B.
The diversity of Phytophthora species associated with the three different plant types
was calculated using the Margalef richness index (d) [
49
], the Shannon diversity index
(H) [
50
] and the evenness index (J) [
51
]. The indices were calculated using Past software,
version 4.03 [52].
Similarity in terms of taxonomic richness among the communities within the three
plant categories was schematized through the use of Venn diagrams [
53
], using GeneVenn
software to generate the diagram (https://www.bioinformatics.org/gvenn/ accessed on
23 June 2023) and reconstructing it in Canva (https://www.canva.com/ accessed on
23 June 2023).
Results of the pathogenicity test were checked for normality, then subjected to analysis
of variance (ANOVA). Significant differences among mean values were determined using

Forests 2023,14, 1515 8 of 24
Fisher’s least significant differences multiple range test (p= 0.05) after one-way ANOVA
using XLSTAT 2008 software (Addinsoft, Paris, France).
3. Results
3.1. Symptomatology
Monitoring surveys conducted in 54 sites distributed in Italy and Slovenia allowed the
occurrence of Phytophthora-related diseases to be detected in several plants typical of the alpine
and subalpine climate. Disease incidence was highest in shrub vegetation, alpine heathlands
and along the mountain riparian systems, ranging from 25 to 100%, with a mortality rate
between 5 and 45% (Table 3). The most impacted ecosystems were heathlands dominated by
common juniper and blueberry, and alder riparian systems (Figure 1). In these ecosystems,
Phytophthora outbreaks showed an epidemic trend with a high mortality rate.
Figure 1.
Overview of aerial Phytophthora disease symptoms observed on: Alnus viridis (
1
), Juniperus
communis (
2
), Rhododendron ferrugineum (
3
)Vaccinium myrtillus (
4
); Alnus cordata (
5
), Alnus glutinosa
(
6
), Alnus viridis (
7
,
8
), Juniperus communis (
9
,
10
), Ilex aquifolium (
11
), Lycopodium clavatum (
12
), Pinus
mugo (
13
), Populus tremula (
14
), Salix caprea (
15
), Taxus baccata (
16
), Rhododendron spp. (
17
–
19
)
,
Vac-
cinium spp. (
20
–
22
); Alnus spp. (
23
–
25
), Ilex aquifolium (
26
), Fagus sylvatica (
27
) and Salix caprea (
28
).
On the left, starting from the top, colony morphology of: Phytophthora acerina,P. alpina,P. bilorbang,
P. cactorum,P. cambivora,P. chlamydospora,P. gonapodyides, P. hedraiandra,P. idaei, P. ilicis,P. kelmanii,
P. plurivora,P. pseudocryptogea,P. pseudogregata,P. pseudosyringae,P. psychrophila,P. rosacearum and
P. syringae after 7 days of growth at 20 ◦C on CA in the dark.

Forests 2023,14, 1515 9 of 24
Table 3. Symptoms observed on each plant host and disease incidence/mortality rate estimated.
Plant Species Symptoms Observed Disease
Incidence (%)
Mortality
Rate (%)
Acer pseudoplatanus Bleeding cankers, inner bark necrosis nd * nd
Acer monspessulanum Bleeding cankers, inner bark necrosis nd nd
Alnus cordata Foliar necrosis, shoot blight, wilting nd nd
Alnus glutinosa Bleeding cankers, shoot blight, wilting nd nd
Alnus incana Bleeding cankers, inner bark necrosis, shoot blight 55–100 15–35
Alnus viridis
Bleeding cankers, shoot blight, wilting, foliar necrosis, sudden death
80–100 17–42
Betula pendula Bleeding cankers, inner bark necrosis nd nd
Calluna vulgaris Shoot blight, wilting, sudden death nd nd
Erica carnea Shoot blight, wilting nd nd
Fagus sylvatica Bleeding cankers, inner bark necrosis nd nd
Fragaria vesca Foliar necrosis, wilting nd nd
Fraxinus excelsior Bleeding cankers, inner bark necrosis
Genista corsica Wilting, sudden death nd nd
Ilex aquifolium Foliar necrosis, wilting, bleeding cankers, inner bark necrosis 100 5
Juniperus communis Shoot blight, wilting, sudden death 25–100 3–40
Laburnum alpinum Bleeding cankers, inner bark necrosis 80 30
Larix decidua Shoot blight, wilting nd nd
Lonicera alpigena Foliar necrosis, wilting nd nd
Lycopodium clavatum Foliar necrosis, wilting, sudden death nd nd
Pinus mugo Wilting, shoot blight, sudden death 20–60 5–25
Populus tremula Foliar necrosis, shoot blight, wilting 100 -
Quercus pubescens Bleeding cankers 100 27
Rhododendron ferrugineum
Bleeding cankers, shoot blight, wilting, foliar necrosis, sudden death
42–84 12–26
Rhododendron hirsutum Shoot blight nd nd
Rubus idaeus Foliar necrosis nd nd
Salix alpina Shoot blight, wilting, sudden death 17 5
Salix atrocinerea Bleeding cankers, inner bark necrosis nd nd
Salix caprea Foliar necrosis, bleeding cankers, inner bark necrosis, epicormic
shoots 68–83 14–34
Sorbus aria Bleeding cankers, inner bark necrosis nd nd
Sorbus aucuparia Bleeding cankers, inner bark necrosis, shoots blight 90 30
Taxus baccata Shoot blight, wilting nd nd
Vaccinium myrtillus Foliar necrosis, fruit rot, shoot blight. sudden death 30–80 10–45
Vaccinium vitis-idaea Foliar necrosis, fruit rot, shoot blight, sudden death nd nd
* nd = Not determined.
Many of the aerial Phytophthora symptoms observed were new and involved various
plant organs such as leaves (moist necrotic lesions), fruit (rot), twigs (wilting and shoot
blights). Moreover, on tree and shrub species stem and branches extensive bleeding cankers
were observed (Figure 1). Cankers and necrosis progressively girdled the circumference of
the branch, causing partial or total death of the crown.

Forests 2023,14, 1515 10 of 24
On shrubs and heath formations, the disease was initially observed in small areas and
progressively spread in a concentric manner affecting more plant species (Figure 1).
3.2. Aetiology
Isolations performed on 397 samples yielded a total of 360 Phytophthora isolates. Based
on morphological features and ITS sequence data, 17 known Phytophthora species were
identified, namely: P. pseudosyringae (201 isolates), P. plurivora (54), P. gonapodyides (21),
P. ilicis (20), P. alpina (17), P. acerina (11), P. cactorum (7), P. pseudocryptogea (6), P. cambivora (5),
P. idaei (4), P. psychrophila (3), P. bilorbang (2), P. chlamydospora (2), P. hedraiandra (1), P. kel-
manii (1), P. rosacearum (1) and P. syringae (1).
In addition, three isolates obtained from necrotic tissues of Alnus viridis,Juniperus
communis and Rhododendron ferrugineum could not be assigned to any known Phytophthora
species and are therefore described here as Phytophthora pseudogregata sp. nov.
The assemblage and distribution of Phytophthora species was very variable among hosts
and geographic areas. The 33 plant species monitored were divided into three main cate-
gories: small trees (Table 4), shrubs/heathland species (Table 5) and herbaceous/perennial
plant species (Table 6).
The most common and widespread Phytophthora species detected in this study was
P. pseudosyringae. This species was isolated from 25 out of the 33 hosts, in 36 sites distributed
in all monitored geographic regions. Together with P. cactorum, it is the only species detected
in all three types of hosts, while the other Phytophthora species were isolated from only
one or two types (Figure 2). Phytophthora plurivora was the second most-isolated species,
obtained from 12 hosts in 24 sites.
Phytophthora pseudosyringae and P. plurivora were the most frequently isolated species
in NE Italy and Slovenia (Figure 2). In addition to these two species, some species belonging
to clade 1, such as P. alpina and P. cactorum, were frequently isolated from different hosts
in the NE Alps. In the mountainous areas of Sardinia, in addition to P. pseudosyringae,
other two species P. ilicis and P. psychrophila belonging to clade 3 were constantly isolated
(Figure 2).
Table 4.
Number of Phytophthora isolates obtained from the different plant hosts. In brackets
the number of sites for each Phytophthora species: Phytophthora acerina (ACE), P. cactorum (CAC),
P. cambivora (CAM), P. gonapodyides (GON), P. idaei (IDA), P. ilicis (ILI), P. kelmanii (KEL), P. plurivora
(PLU), P. pseudosyringae (PSS) and P. psychrophila (PSY).
Tree Species
Phytophthora Isolates (Number of Sites)
ACE CAC CAM GON IDA ILI KEL PLU PSC PSS PSY
Acer pseudoplatanus - - - - - - - 2 (2) - - -
A. monspessulanum - - - - - - - - - 2 (1) * -
Alnus cordata - - - - - - - - - 1 (1) * -
Alnus glutinosa - - - 2 (2) - - - - - 3 (2) -
Alnus incana - 1 (1) 1 (1) * - 2 (1) * - 1 (1) * 5 (3) - - -
Betula pendula - - - - - - - 2 (1) - - -
Fagus sylvatica - - - - - - - - - 3 (3) -
Fraxinus excelsior 4 (3) * - - - - - - 5 (4) - 1 (1) * -
Ilex aquifolium - - - 2 (1) * - 20 (4) - - - 1 (1) -
Laburnum alpinum - - 2 (1) * - - - - - - - -
Larix decidua - 1 (1) - - - - - - - 1 (1) * -
Populus tremula - - - - - - - - 2 (1) * 16 (4) * -
Quercus pubescens - - - - - - - - - 1 (1) 3 (1)
Salix caprea 2 (2) * - - - - - - 12 (6) * - 3 (3) * -
Sorbus aria - 1 (1) * - 2 (1) * - - - - - 1 (1) * -
Sorbus aucuparia - - 2 (1) * - - - - 2 (2) * - 2 (2) * -
Taxus baccata - - - - - - - - - 1 (1) * -
* New host–pathogen associations.

Forests 2023,14, 1515 11 of 24
Table 5.
Number of Phytophthora isolates obtained from the different plant hosts. In brackets the
number of sites for each Phytophthora species: Phytophthora acerina (ACE), P. alpina (ALP), P. cactorum
(CAC), P. chlamydospora (CHL), P. hedraiandra (HED), P. plurivora (PLU), P. pseudocryptogea (PSC),
P. pseudogregata (PSG), P. pseudosyringae (PSS), P. rosacearum (ROS) and P. syringae (SYR).
Shrub and Heathland
Species
Phytophthora Isolates (Number of Sites)
ACE ALP BIL CAC CHL GON HED PLU PSC PSG PSS ROS SYR
Alnus viridis -8 (4) -1 (1) * -1 (1) * 1 (1) * 4 (2) * 2(1) 1 (1) * 22 (8) - -
Calluna vulgaris ----------3 (2) * - -
Erica arborea sbsp. alpina ----------2 (1) * - -
Genista corsica ----------1 (1) * - -
Juniperus communis 1 (1) * -2(1) * - - 7 (1) * -7 (4) * -1(1) * 20 (8) 1 (1) * -
Lonicera alpigena -1 (1) * -------- - --
Pinus mugo -------3 (3) * 2(1) * -9 (7) * - -
Rhododendron ferrugineum 1 (1) * ----1 (1) * -6 (3) * -1(1) * 24 (10) * - -
Rhododendron hirsutum ----------2 (1)* - -
Salix alpina ----2 (1) * - - 1 (1) * - - 1 (1) * - -
Salix atrocinerea ---------- - -1 (1) *
Vaccinium myrtillus 3 (2) * 7 (3) * ---4 (2) * -5 (3) * - - 67 (14) - -
Vaccinium vitis-idaea -1 (1) * ---2 (1) * ----2 (2) - -
* New host–pathogen associations.
Table 6.
Number of Phytophthora isolates obtained from the herbaceous plant hosts. In brackets
the number of sites for each Phytophthora species: Phytophthora cactorum (CAC), P. idaei (IDA) and
P. pseudosyringae (PSS).
Herbaceous and Perennial Species Phytophthora Isolates (Number of Sites)
CAC IDA PSS
Fragaria vesca 3 (2) - -
Lycopodium clavatum - - 12 (1) *
Rubus idaeus - 2 (1) -
* New host–pathogen associations.
Figure 2.
Isolation frequency and distribution of the 7 most common Phytophthora species isolated in
this study.

Forests 2023,14, 1515 12 of 24
As regards the distribution within Phytophthora clades, clade 6 is the most represented
in terms of species (five species) followed by clade 1 (4), clade 3 (3) and clade 8 (3). Only one
or two species were obtained for clades 2 and 7. Overall, 56 new host–pathogen associations
were detected (Tables 4–6).
3.3. Structure and Diversity of Phytophthora Communities
The diversity indices of the Phytophthora assemblages detected in the subalpine veg-
etations varied among the three categories of hosts, but in general they displayed high
diversity and richness and moderate evenness, with the exception of the shrub Phytophthora
community dominated by P. pseudosyringae (Table 7).
Table 7.
Values of the diversity indices, Shannon diversity index (S), Margalef index (d) and Pielou
Evenness (J) of Phytophthora populations from three different plant communities.
Plant Types Taxa Shannon Index (H) Margalef (d) Pielou Evenness (J)
Tree species 11 1.867 2.158 0.588
Shrub species 13 1.348 2.227 0.296
Herbaceous species 3 0.804 0.706 0.745
Tree and shrub species displayed the highest number of taxa and Shannon index (H)
values. As regards the degree of similarity between the three Phytophthora communities,
the Jaccard similarity index (Jc) was variable between 0.11 and 0.20. Only two Phytophthora
species, P. pseudosyringae and P. cactorum, were isolated from all host groups. Relationships
among the three categories of hosts are shown in Figure 3.
Figure 3.
Venn diagrams illustrating the number of unique and shared Phytophthora species among the
three categories of plant species. The outer numbers indicate values of the Jaccard similarity coefficient.
3.4. DNA Phylogeny
Phylogenetic relationships among the Phytophthora isolates obtained in this study were
elucidated using ITS sequences (Figure 4). In particular, the 20 isolates included in the
phylogenetic analysis were distributed in 18 terminal clades, 17 of which belong to formally
described species (Figure 4). Instead, three isolates clustered together in a separate and well-

Forests 2023,14, 1515 13 of 24
supported terminal clade (ML bootstrap = 100%) representing a previously unrecognized
species closely related to P. gregata, which is described here as Phytophthora pseudogregata sp.
nov. (Figure 4).
Figure 4.
Maximum likelihood tree obtained from internal transcribed spacer (ITS) sequences of
Phytophthora species representative of the 12 clades. Data are based on the General Time Reversible
model. A discrete Gamma distribution was used to model evolutionary rate differences among sites.
The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
Bootstrap support values in percentage (1000 replicates) are given at the nodes. Ex-type cultures are
in bold, and isolates obtained in this study in red.
To resolve the phylogenetic position of P. pseudogregata within subclade 6b, a concate-
nated nuclear and mitochondrial dataset (the length of the final alignment was 2129 bp)
was analysed. Individual gene phylogenies revealed no major conflicts, thus indicating
that the three loci (ITS, Btub and cox1) could be combined. The ML analysis resolved the

Forests 2023,14, 1515 14 of 24
positions of all formally described Phytophthora species in subclade 6b, accommodating
the isolates P. pseudogregata in a terminal clade sister to P. gibbosa (Figure 5). Phytophthora
pseudogregata is separated by the two closely related species, P. gregata and P. gibbose, by
three, two, and 18 bp and by eight, three, and 17 bp in ITS, Btub, and cox1 loci, respectively.
Figure 5.
Maximum likelihood tree obtained from concatenated ITS, Btub and cox1 sequences of the
Phytophthora species belonging to subclade 6b. Data are based on the General Time Reversible model.
A discrete Gamma distribution was used to model evolutionary rate differences among sites. The tree
is drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap
support values in percentage (1000 replicates) are given at the nodes. Ex-type cultures are in bold
and isolates of the study in red.

Forests 2023,14, 1515 15 of 24
3.5. Taxonomy
Phytophthora pseudogregata Bregant, Ogris, Meli and Linaldeddu sp. nov.
MycoBank: MB849354
Etymology: the name refers to the morphological similarity to Phytophthora gregata.
Holotype: CBS H-25226
Host/distribution: Alnus viridis,Juniperus communis and Rhododendron ferrugineum
with foliar necrosis and shoot blight symptoms in Italy and Slovenia.
Description: Sporangia were produced on CA plugs flooded in unsterile pond wa-
ter after 36–72 h of incubation at 25
◦
C on simple sporangiophores. Sporangia were
persistent, mostly nonpapillate (80%), rarely semipapillate (20%), from ovoid to obpyri-
form, sometimes ellipsoid, borne terminally on unbranched sporangiophores, average
50.3 ±6.5 ×29.9 ±3.8 µm
(total range 32.1–65.1
×
22.1–38.4
µ
m), with a length/breadth
ratio of 1.7
±
0.2 (n= 50) (Figure 6d–g). Zoospores were abundantly produced in liquid
cultures after 24–36 h at 25
◦
C in the dark (Figure 6h). Sporangia proliferated, usually
externally and rarely internally, in both a nested and extended way (Figure 6i–k). Hyphal
swellings were not formed on solid agar and rarely in pond water, they were globose to
subglobose, mostly intercalary catenulate, rarely terminal (Figure 6l–m). Chlamydospores
were not observed. All isolates produced gametangia in single culture on carrot agar
after 7–10 days at 20
◦
C in the dark. Oogonia were smooth-walled, borne mainly ter-
minally, with an average diameter of 33.2
±
3.4. Oospores were spherical and usually
aplerotic and
29.0 ±3.7 µm
in diameter. Antheridia were mostly amphigynous (58%),
less frequently paragynous (42%) hyaline, rounded, club-shaped, or irregular: average
16.2 ±2.7 ×12.5 ±2.2 µm (Figure 6o–r).
Figure 6.
Colony morphology of Phytophthora pseudogregata after 7 days growth at 20
◦
C on PDA (
a
),
MEA (
b
), and CA (
c
). Persistent sporangia, nonpapillate (
d
,
e
) semipapillate (
f
,
g
), releasing of
zoospores (
h
), external (
i
) and internal (
j
,
k
) proliferations; intercalary (
l
) and terminal hyphal
swellings (
m
); mycelia (
n
). Oogonia with amphygynous (
o
,
p
) and paragynous antheridia (
q
,
r
).
Scale bars = 20 µm.

Forests 2023,14, 1515 16 of 24
Cultural characteristics: colony growth pattern cottony on PDA with an irregular
border, with an indistinct pattern on MEA and CA. On PDA, growth was slow, whereas on
MEA and CA, colonies reached a diameter of 55 and 70 mm in 7 days at 23
◦
C, respectively.
Cardinal temperatures for growth: minimum <2
◦
C, maximum 32
◦
C, and optimum
23
◦
C. Isolates failed to grow at 34
◦
C, and mycelium did not resume growth when plates
were moved to 20 ◦C.
Material examined: ITALY: Borso del Grappa, isolated from a necrotic shoot of Junipe-
rus communis, 13 June 2022, collected by Letizia Meli, isolated by Carlo Bregant, HOLOTYPE
CBS H-25226, a dried culture on CA, culture ex-holotype CB234 = CBS 149859. ITALY: San
Nicolòdi Comelico, isolated from necrotic leaves of Rhododendron ferrugineum, 3 July 2021,
collected and isolated by C. Bregant (isolate CB308). SLOVENIA: Bohinj, isolated from a
necrotic branch of Alnus viridis, 7 October 2021, collected by C. Bregant and Nikica Ogris
and isolated by C. Bregant (isolate CB366).
Notes: Phytophthora pseudogregata belongs to subclade 6b. The closest species are P. gre-
gata and P. gibbosa, from which it differs through a combination of unique morphological
features (Table 8) and sequence data such as sporangia size and proliferation, oogonia and
antheridia shapes and cardinal temperature values, as well as a total of 23 (P. gregata) and
28 (P. gibbosa) fixed nucleotide differences in the ITS, Btub, and cox1 sequences.
Table 8.
Morphological features, morphometric data and temperature–growth relationship of Phy-
tophthora pseudogregata and closely related species in subclade 6b.
P. pseudogregata P. gregata P. gibbosa
Number of isolates examinated 3[54] [54]
Sporangia
Ovoid to obpyriform,
sometimes ellipsoid,
nonpapillate, some
semipapillate
Ovoid, limoniform, obpyriform,
nonpapillate Ovoid, ellipsoid, nonpapillate,
some semipapillate
Length ×breadth mean (µm) 50.3 ±6.5 ×29.9 ±3.8 51.0 ±13.8 ×30.5 ±5.9 48.8 ±9.6 ×30.8 ±5.4
Total range 32.1–65.1 ×22.1–38.4 25.7–102.3 ×14.8–50.7 24.8–71.1 ×17.4–48.0
Length/Breadth ratio 1.7 ±0.2 1.67 ±0.32 1.58 ±0.15
Proliferation Mostly external, sometimes
internal, mostly extended and
rarely nested
Internal extended and nested,
never external, sporangiophore
partly branching inside empty
sporangium
Internal extended, external,
never nested
Hyphal swellings
Globose to subglobose, mostly
intercalary catenulate, rarely
terminal
Globose, elongated, angular,
partly catenulate Subglobose, elongated, never
catenulate
Chlamydospores Not observed Not observed Not observed
Breeding system Homotallic Homothallic or self-fertile Homotallic
Oogonia Smooth Smooth Ornamented, smooth
Mean diameter (µm) 33.2 ±3.4 36.8 ±4.1 38.1 ±5.4
Diameter range (µm) 26.9–41.6 23.9–50.9 27.0–49.9
Oospores Aplerotic Usually aplerotic Always aplerotic
Mean diameter (µm) 29.1 ±3.7 31.6 ±4.0 31.4 ±4.6
Total range (µm) 20.5–37.8 21.4–45.3 18.9–39.4
Wall thickness (µm) 2.18 ±0.71 2.65 ±0.81 3.17 ±0.69
Antheridia
Mostly amphygynous (58%),
less frequently
paragynous (42%) Mostly paragynous Amphigynous
Length ×breadth mean (µm) 16.2 ±2.7 ×12.5 ±2.2 17.1 ±3.0 ×11.0 ±1.8 13.6 ±2.4 ×14.0 ±2.0
Total range (µm) 11.7–23.3 ×9.0–17.9 10.6–24.9 ×7.6–17.8 10.6–24.9 ×7.6–17.8
Maximum temperature (◦C) 32 32.5 ≤35 32.5 ≤35
Optimum temperature (◦C) 23 25 30

Forests 2023,14, 1515 17 of 24
3.6. Pathogenicity
All Phytophthora species proved to be pathogenic on Juniperus communis. At the end of
the experimental period, inoculated seedlings showed dark brown inner bark lesions that
spread up and down from the inoculation point (Figure 7).
Among the different species assayed, the length of the necrotic lesion differed significantly
(Table 9). The lesions caused by P. pseudosyringae were significantly larger than those caused
by other species (Table 9). Lesions caused by P. pseudosyringae,P. plurivora and P. acerina
progressively girdled the twigs causing shoot blight, browned foliage and wilting symptoms.
Control seedlings, inoculated with sterile PDA plugs, remained symptomless; in only
two twigs, a small light brown discoloration was observed restricted to the inoculation point.
All eight Phytophthora species were successfully re-isolated from the necrotic inner
bark lesions of all seedlings, thus fulfilling Koch’s postulates. No Phytophthora or other
fungal isolates were obtained from control plants.
Figure 7.
Symptoms observed on common juniper twigs 30 days after inoculation with Phytoph-
thora acerina (
a
,
b
), P. bilorbang (
c
,
d
), P. gonapodyides (
e
,
f
), P. plurivora (
g
,
h
), P. pseudocryptogea (
i
,
j
),
P. pseudogregata (k,l), P. pseudosyringae (m,n) and P. rosacearum (o,p). Control (q,r).

Forests 2023,14, 1515 18 of 24
Table 9.
Mean lesion length
±
standard deviation caused by each Phytophthora species on common
juniper twigs.
Species Isolate Mean Lesion
Length (cm) *
Wilted
Foliage
Re-Isolation
(%)
P. acerina CB222 1.4 ±0.2 e yes 100
P. bilorbang CB600 1.1 ±0.7 f no 80
P. gonapodyides CB367 1.4 ±0.3 de no 100
P. plurivora CB358 1.9 ±0.2 b yes 100
P. pseudocryptogea CB482 1.7 ±0.3 c no 100
P. pseudogregata CBS149859 1.7 ±0.2 cd no 100
P. pseudosyringae CB303 2.3 ±0.4 a yes 100
P. rosacearum CB481 1.3 ±0.2 ef no 100
Control - 0.2 ±0.1 g no -
LSD critical value 1.90
*Values with the same letter do not differ significantly at p= 0.05, according to LSD multiple range test.
4. Discussion
This study represents the most comprehensive investigation to date on aerial diseases
caused by Phytophthora species on mountain vegetation in Italy and Slovenia. The results
obtained have allowed us to clarify both symptomatology and aetiology of the emerging
pathosystems affecting mountain and subalpine formations. The progressive spread of
several airborne Phytophthora species is causing the destruction of vast ecosystems and
compromising the biodiversity of these ecologically fragile habitats.
Based on combined sequence data and micromorphological features, 18 Phytophthora
species belonging to six out the 12 major Phytophthora phylogenetic clades were iden-
tified from a collection of 397 symptomatic samples collected from 33 herbaceous and
woody hosts. These included: P. acerina,P. alpina,P. bilorbang,P. cactorum,P. cambivora,
P. chlamydospora,P. gonapodyides,P. hedraiandra,P. idaei,P. ilicis,P. plurivora,P. pseudocryptogea,
P. pseudosyringae,P. psychrophila,P. rosacearum and P. syringae. In addition, three isolates
described here as Phytophthora pseudogregata sp. nov. were isolated and characterized.
The most frequently isolated Phytophthora species belong mainly to clades 1 and 3.
These species are characterized by the ability to produce caducous sporangia useful for
aerial infections [
1
]. Furthermore, the relatively low cardinal temperatures for growth
suggest that these species have a great potential to threaten mountain vegetations [
14
,
31
,
55
].
In particular, in Northeast Italy a higher number of species belonging to the ITS clade 1
was isolated (P. alpina,P. cactorum,P. hedraiandra and P. idaei), while in Sardinia, clade 3
was dominant, with three species, P. pseudosyringae,P. ilicis and P. psychrophila. Overall,
P. pseudosyringae (clade 3) was the most frequent species in terms of number of hosts infected
and distribution among sites. Two hundred and one out of the 360 isolates obtained in this
study belonged to this species. In particular, P. pseudosyringae have been detected in 36 sites
and 25 hosts of all three plant categories investigated: trees, shrubs and herbaceous plants
(17 new host–pathogen associations). The wide spread of P. pseudosyringae in different
mountain and subalpine formations and its involvement in several new diseases highlight
the polyphagous nature of this invasive pathogen and its aerial lifestyle. This agrees with
previous studies conducted in mountain environments in Asia, Europe, North and South
America [
14
,
32
,
34
,
55
–
61
]. Phytophthora pseudosyringae is the key species in the aetiology of
aerial infections detected in high-altitude shrubs and heaths such as blueberry, dwarf pine,
juniper, rhododendron and alpine willow formations; these shrubs are characterized by
creeping behaviour with very limited heights above the ground, this habitus could favour
the attack of Phytophthora sporangia and zoospores. The attacks of P. pseudosyringae on
Vaccinium myrtillus (leaf necrosis and shoot blight) were particularly severe, confirming the
susceptibility of this small shrub as previously reported in Ireland [
33
,
62
]. Many aspects
regarding the infectivity and survival of P. pseudosyringae sporangia in the infected tissues
fallen to the ground in subalpine areas remain to be clarified. At the same time the ability
of oospores to persist for years and their infectivity in environments where the pathogen is

Forests 2023,14, 1515 19 of 24
subjected to extreme low temperatures need further investigations. Probably the survival
of this species in cold habitats is guaranteed by the production of very large and thick
wall chlamydospores. In fact, unlike what was reported in previous studies and in the
original description, all isolates of P. pseudosyringae examined produced a large amount
of globose chlamydospores on CA both in solid and liquid culture. The chlamydospores
were mainly terminal and 76.6
±
22.02 (range 39.9–102.3, n= 25)
µ
m in diameter. Based
on the wide variation in morphological characters found in this study, the description of P.
pseudosyringae needs to be redefined. Undoubtedly, increased inoculum in the litter due
to the diseased fallen leaves not only could represent an increased risk of outbreaks but
also a faster disease progression in these habitats [Bregant & Linaldeddu, unpublished].
In the pathogenicity test, P. pseudosyringae shows high aggressivity on common juniper,
producing wood necrosis and shoot blight after four weeks from the inoculation.
The other two species in clade 3 were isolated only in the mountain area of Sardinia.
Phytophthora psychrophila have been isolated from bleeding cankers of Quercus pubescens,
confirming the affinity of this pathogen towards oak species [
63
]; the geographic distri-
bution and impact of this species is still unknown; there have been a few reports of it in
European and American natural forests and nurseries [
4
,
31
,
64
]. Phytophthora ilicis has been
known for a long time as a specific pathogen of Ilex aquifolium in the mountains of the
Mediterranean basin and a few other areas of Europe and North America [55,65–67].
Four species belonging to subclade 1a have been isolated in the northeastern Alps.
Phytopththora alpina shows the highest ability to survive in extremely cold conditions due
to the low temperature values for growth and the high production of caducous sporangia
and chlamydospores [
14
]; in addition to Alnus viridis, its discovery on three new hosts in
Italy and Slovenia suggest that this recently described species is well adapted to affect
typical alpine and subalpine shrubs. The second most common species in subclade 1a
was P. cactorum, an invasive and polyphagous pathogen widespread from tropical to
temperate climates where it is responsible for severe diseases on agriculture crops and
forest trees [
1
,
29
,
68
]. The occurrence of P. cactorum in cold areas has recently been reported
in Europe and Australia [
4
,
14
,
60
,
69
]. Together, P. pseudosyringae and P. cactorum are the two
species obtained from all three plant types.
In addition to the numerous new host–pathogen associations (Tables 4–6), some
species detected such as P. hedraiandra and P. idaei are reported for the first time in natural
ecosystems in Europe. Previous studies have ascertained the involvement of these two
pathogens in root and foliar disease in agriculture and ornamental nurseries; P. idaei appears
restricted to the genus Rubus [
70
], while P. hedraiandra has a wider range of ornamental
hosts [
71
–
75
]. Although, in the original description P. idaei is reported to have persistent-
sporangia, the Italian isolates obtained in this study showed a moderate production of
caducous sporangia.
The second most common species obtained in this study was P. plurivora. Isolates of
this species were obtained from 54 symptomatic samples of 12 plant species including eight
new hosts. Phytophthora plurivora resides in clade 2 and is common in forest ecosystems
of Central Europe; from a recent population study it is considered to be originally of this
continent and spread to others by human activities [
76
]. This agrees with the results of this
and previous studies [
8
,
14
] given the wide distribution of this pathogen in various extreme
and nonhumanized natural environments. While the distribution and impact of P. plurivora
is well studied, little is known about its closely related species, P. acerina. To date, this
species appears widespread in agricultural systems, nurseries, forests and ornamental trees
in northern Italy and Sardinia, and much rarer worldwide [
4
,
77
–
79
]. Both P. acerina and
P. plurivora were already known as primary pathogens involved in common and grey alder
decline in Italy [
14
]. Isolates of Phytophthora acerina obtained in this study confirm a single
polymorphism in the ITS region between northern Italy and Sardinia populations [14].
Among the other Phytophthora species isolated in this study, five belong to clade 6,
including the newly described species P. pseudogregata. Clade 6 encompasses species
very common in European forests, such as P. bilorbang and P. gonapodyides and species

Forests 2023,14, 1515 20 of 24
with more limited or still unknown distribution, such as P. amnicola and P. rosacearum [
8
].
Some species in this clade are reported as saprophyte or occasionally weak opportunistic
pathogens [
11
,
54
,
80
,
81
]; the involvement of five species of this clade in the aetiology of aerial
infections on mountain vegetations highlight the ecological versatility of these organisms.
The ability of P. bilorbang,P. gonapodyides and P. pseudogregata to reproduce the symptoms
observed in nature on common juniper suggest their active role in the aetiology of the
emerging disease affecting woody trees in mountain areas.
Phytophthora pseudogregata resides in subclade 6b; it is closely related to P. gregata
and P. gibbosa, from which it can be distinguished by unique morphological features and
sequence data. Phytophthora gregata was originally described in 2011 in Australia in wet
native forests and in Tasmania associated with dying alpine heathland vegetation [
54
,
69
]
and then recently reported in the Czech Republic and Finland [
60
,
82
], whereas P. gibbosa is
known to occur only in Australia associated with dying native vegetation on seasonally
wet sites [54].
Sub-clade 6b is larger and contains several described species (P. amnicola,P. borealis,
P. chlamydospora,P. bilorbang,P. crassamura,P. fluvialis,P. gibbosa,P. gonapodyides,P. gregata,
P. lacustris,P. litoralis,P. megasperma,P. mississippiae,P. moyootj,P. ornamentata,P. pinifolia,
P. pseudogregata,P. riparia and P. thermophila), some not formally described species and a
few hybrids [
83
]. Most of the species in this sub-clade have been described in the last
12 years, the only species known until 2011 were P. gonapodyides and P. megasperma [
1
]. The
majority of species in sub-clade 6b, including P. pseudogregata, have been described in forest
ecosystems, underlining the key role played by natural areas in exploring the biodiversity
of the Phytophthora genus, which currently includes 220 species (Figure 8).
Figure 8.
Number of Phytophthora species described per year since 1997, divided into species isolated
from nurseries and agriculture and forest ecosystems. The graph also reports the progression
of the described species over time (black line). (Source: Scopus, May 2023 and IndexFungorum
May 2023, [1]).
Finally, three species of clade 8 (P. kelmanii,P. pseudocryptogea and P. syringae) and
one from clade 7 (P. cambivora) have been isolated, mainly from stem bleeding cankers of
small trees and shrubs. While P. kelmanii and P. syringae have a very limited distribution,
P. pseudocryptogea is widespread along the Alps. The large range of growth temperatures
and polyphagous nature explain it being widespread in Italian ecosystems spanning from

Forests 2023,14, 1515 21 of 24
Mediterranean areas to the tree line in the Dolomites [
4
,
14
,
78
]. Both mating types of
P. cambivora occurred in the NE Alps (A2 on Alnus incana in Slovenia and A1 on Laburnum
alpinum and Sorbus aucuparia in Italy).
5. Conclusions
In conclusion, the discovery of several emerging Phytophthora diseases in the canopy
of subalpine vegetations in Europe is of particular concern and underlines the need to
further extend research into these environments to assess the full diversity of Phytophthora
clades and species and the factors driving the emergence and diffusion of these invasive
pathogens. Studying Phytophthora communities on necrotic leaves naturally fallen, would
be useful to evaluate host specificity, geographic distribution and survival strategies of the
main Phytophthora species detected in this study.
A survey is currently in progress to map the distribution of P. pseudogregata in Alpine
habitats and to establish the natural host range of this new taxon.
Author Contributions:
B.T.L., C.B. and N.O. conceptualization; B.T.L., C.B., G.R., L.M. (Letizia Meli),
N.O., N.S. and A.B. field survey, sample collection and assay; B.T.L., C.B., G.R. and L.M. (Letizia
Meli) data analysis; B.T.L., L.M. (Lucio Montecchio), L.M. (Lucia Maddau), B.P. and N.O. funding
acquisition; C.B. draft writing; B.T.L., L.M. (Lucio Montecchio), L.M. (Lucia Maddau), A.B., B.P. and
N.O. review and manuscript editing. All authors have read and agreed to the published version of
the manuscript.
Funding:
This research was partially funded by grant number DOR2305524/2023 “Monitoraggio
dei marciumi radicali da Phytophthora negli ecosistemi forestali Italiani”, by the Land Environment
Resources and Health (L.E.R.H.) doctoral course (University of Padova), by Fondo di Ateneo 2020, an
internal funding by the University of Sassari and by Slovenian Research Agency (Research Program
P4-0107 Forest Biology, Ecology and Technology), Slovenian Ministry of Agriculture, Forestry and
Food (Public Forestry Service).
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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