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Citation: Fiorenza, A.; Aiello, D.;
Costanzo, M.B.; Gusella, G.; Polizzi,
G. A New Disease for Europe of Ficus
microcarpa Caused by
Botryosphaeriaceae Species. Plants 2022,
11, 727. https://doi.org/10.3390/
plants11060727
Academic Editors: Dirk Janssen and
Yasser Sobhy Ahmed Nehela
Received: 17 February 2022
Accepted: 6 March 2022
Published: 9 March 2022
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4.0/).
plants
Article
A New Disease for Europe of Ficus microcarpa Caused by
Botryosphaeriaceae Species
Alberto Fiorenza †, Dalia Aiello †, Mariangela Benedetta Costanzo, Giorgio Gusella * and Giancarlo Polizzi
Department of Agriculture, Food and Environment (Di3A), University of Catania, Via S. Sofia 100,
95123 Catania, Italy; alberto.fiorenza@phd.unict.it (A.F.); dalia.aiello@unict.it (D.A.);
benedettacst@gmail.com (M.B.C.); gpolizzi@unict.it (G.P.)
*Correspondence: giorgio.gusella@phd.unict.it
† These authors contributed equally to this work.
Abstract:
The Indian laurel-leaf fig (Ficus microcarpa) is an important ornamental tree widely dis-
tributed in the urban areas of Italy. Surveys conducted in 2019 and 2020 on several tree-lined streets,
squares, and public parks in Catania and Siracusa provinces (Sicily, southern Italy) revealed the
presence of a new disease on mature trees. About 9% of approximately 450 mature plants showed
extensive branch cankers and dieback. Isolations from woody tissues obtained from ten symptomatic
plants consistently yielded species belonging to the Botryosphaeriaceae family. The identification of
the recovered fungal isolates was based on a multi-loci phylogenetic (maximum parsimony and
maximum likelihood) approach of the ITS, tef1-α, and tub2 gene regions. The results of the analyses
confirmed the presence of three species: Botryosphaeria dothidea,Neofusicoccum mediterraneum, and
N. parvum
. Pathogenicity tests were conducted on potted, healthy, 4-year-old trees using the mycelial
plug technique. The inoculation experiments revealed that all the Botryosphaeriaceae species identified
in this study were pathogenic to this host. Previous studies conducted in California showed similar
disease caused by Botryosphaeriaceae spp., and the pathogenic role of these fungi was demonstrated.
To our knowledge, this is the first report of Botryosphaeriaceae affecting Ficus microcarpa in Europe.
Keywords: canker; dieback; Indian laurel-leaf fig; Ficus microcarpa;Botryosphaeriaceae; phylogeny
1. Introduction
Ficus microcarpa, commonly known as Chinese or Malayan banyan, Indian laurel-leaf
fig, and curtain fig, is a widely distributed evergreen ornamental species belonging to the
family Moraceae, native to Ceylon, India, southern China, the Ryukyu Islands, Australia,
and New Caledonia [
1
]. It is considered one of the most common urban trees in warm
climates worldwide [
2
]. Moreover, F. microcarpa is also well known as an invader species
due to its ability to grow in inhospitable places, its large fruit production, and its numerous
dispersal agents (birds, bats, rodents, and others) [
3
]. Many Ficus spp. were introduced
in southern Italy as ornamental species; they are now common in many urban areas and
viewed as an important form of historical heritage [
4
]. Parks and gardens in urban areas
are of significant value for all people living their daily lives in the cities. Urban trees have a
positive impact on reducing heat, providing a convenient shelter, reducing wind velocity,
and increasing the aesthetic value of the landscape [
5
–
7
]. In addition, most people living in
cities deal with schedules, work, appointments, meetings, etc., and urban parks and open
spaces positively affect mental health [
8
]. Thus, it is important not to underestimate the
health of urban trees.
According to Fungal Database 53 records of fungus association with this host have been
reported worldwide [
9
]. Among these, particular attention is given to species belonging to
Botryosphaeriaceae. In fact, diseases caused by Botryosphaeriaceae are drawing the attention
of the researchers worldwide, since they are a significant threat to many crops, especially
in Mediterranean climates [
10
]. Botryosphaeriaceae include a large group of diverse fungal
Plants 2022,11, 727. https://doi.org/10.3390/plants11060727 https://www.mdpi.com/journal/plants
Plants 2022,11, 727 2 of 14
species, distributed all over the world. These fungi are well known as plant pathogens,
endophytes, and saprophytes of woody hosts [
11
]. Due to their role as plant pathogens,
these fungal species have been studied for a long time, and their impact on forestry and
agricultural production is well known [
12
]. Botryosphaeriaceae induce severe symptoms,
such as branch, shoot, and trunk cankers, and blight fruits and leaves.
Botryosphaeriaceae disease studies on Ficus spp., including the cultivated common
fig (
F. carica
), have been published worldwide, showing that Botryosphaeriaceae and Dia-
porthaceae spp. are involved in complex diseases [
13
–
29
]. Botryosphaeriaceae cause polyetic
epidemics (2–3 cycles per season); thus, the progress of epidemics may extend for several
years [
10
]. In addition, Botryosphaeriaceae, characterized by a wide host range, can easily
jump from one host to another; this is particularly evident in Mediterranean landscapes,
where different crops are cultivated nearby [
10
]. Especially in the case of urban environ-
ments, it must be remembered that dangerous situations are related to the health status
of the trees. Therefore, it is important to monitor the health of trees before they become
hazardous [
30
]. Surveys conducted in the metropolitan area of Catania and Siracusa (Sicily),
during 2019 and 2020 revealed many F. microcarpa distributed among numerous metropoli-
tan areas, including gardens, public parks, tree-lined streets, and squares, showing severe
symptoms of branch cankers and dieback. The aims of this study were to (i) investigate the
etiology of the disease by (ii) characterizing the fungal isolates recovered from diseased
trees based on a multi-loci phylogenetic analysis and (iii) assess their pathogenicity.
2. Results
2.1. Surveys and Fungal Isolations
Ficus microcarpa growing in a wide range of site conditions (tree-lined streets, gardens,
public parks, and squares) have suffered a widespread dieback in Catania and Siracusa
provinces. In the public areas where the research was conducted, more than 40 mature
F. microcarpa
trees (20 to 50 years old) showed cankered twigs and branches on approxi-
mately 450 plants. The trees still appeared green in part of the canopy, although this was
accompanied by parts of branches and shoots that were defoliated and dead (Figures 1A–D
and 2A). Sometimes, it was possible to observe new twigs growing below the damaged
branches (Figure 1C). The sample consisted of large portions of branches showing severe
internal wood discolouration, including of sapwood and the heartwood (Figure 2B–F). Of-
ten, the bark appeared cracked and split along the branches (Figure 2), and internal cankers
were sharply demarcated from adjacent, healthy wood (Figure 2B–F). Isolations frequently
(>70%) yielded Botryosphaeriaceae-like fungi, characterized, as reported by Slippers and
Wingfield [
11
], by a ‘fluffy’ mycelium, either white-to-creamy, pigmented ‘greenish brown’,
or gray-to-gray-black. Moreover, with lower frequencies, colonies of Eutypella-like species
were also isolated from symptomatic tissues.
Plants 2022,11, 727 3 of 14
Plants 2022, 11, x FOR PEER REVIEW 3 of 15
Figure 1. Symptoms of Botryosphaeriaceae disease observed in urban areas on F. microcarpa. (A) Dis-
eased (left) and healthy (right) plants. (B–D) Defoliation and shoot dieback all over the canopy. (C)
New twigs growing below the dead shoots.
Figure 2. Internal symptoms. (A) Dead branch showing cracking of the outer layers of the bark (up-
per), healthy branch (lower). (B–F) Internal cankers and bark cracked along the branch with dis-
eased tissue sharply demarcated from adjacent, healthy wood. Scale bars: (B) = 15 cm; (C) = 50 cm;
(D–F) = 20 cm.
Figure 1.
Symptoms of Botryosphaeriaceae disease observed in urban areas on F. microcarpa.
(A) Diseased
(left) and healthy (right) plants. (
B
–
D
) Defoliation and shoot dieback all over the
canopy. (C) New twigs growing below the dead shoots.
Plants 2022, 11, x FOR PEER REVIEW 3 of 15
Figure 1. Symptoms of Botryosphaeriaceae disease observed in urban areas on F. microcarpa. (A) Dis-
eased (left) and healthy (right) plants. (B–D) Defoliation and shoot dieback all over the canopy. (C)
New twigs growing below the dead shoots.
Figure 2. Internal symptoms. (A) Dead branch showing cracking of the outer layers of the bark (up-
per), healthy branch (lower). (B–F) Internal cankers and bark cracked along the branch with dis-
eased tissue sharply demarcated from adjacent, healthy wood. Scale bars: (B) = 15 cm; (C) = 50 cm;
(D–F) = 20 cm.
Figure 2.
Internal symptoms. (
A
) Dead branch showing cracking of the outer layers of the bark
(upper), healthy branch (lower). (
B
–
F
) Internal cankers and bark cracked along the branch with
diseased tissue sharply demarcated from adjacent, healthy wood. Scale bars: (
B
) = 15 cm; (
C
) =
50 cm
;
(D–F) = 20 cm.
Plants 2022,11, 727 4 of 14
2.2. Morphological Characterization and Phylogenetic Analysis
The PCR amplification of the ITS region, tef1-
α
, and tub2 generated 577 to 581, 273 to
288, and 422 to 446 bp fragments, respectively. The phylogenetic analyses were performed
using a dataset of the three concatenated loci. The sequences generated in this study were
deposited in GenBank (Table 1). A preliminary comparison of our sequences in GenBank
showed our isolates belonging to the genera Botryosphaeria and Neofusicoccum. The Eutypella-
like species showed high similarity with different Eutypella species submitted to GenBank.
Since these isolates were excluded from the phylogenetic analyses due to their results in the
pathogenicity test, they were identified as Eutypella spp. The phylogenetic analyses were
then conducted only for the Botryosphaeriaceae. The results of the partition-homogeneity
test indicated no (p= 1.00) significant differences in the three-gene dataset. The MP
analysis of the combined dataset showed that of 2921 total characters, 391 were parsimony-
informative, 220 were parsimony-uninformative, and 2310 were constant. In total, 100 trees
were retained. Tree length was equal to 1098, CI = 0.707, RI = 0.912,
RC = 0.644
. The best-fit
model of nucleotide evolution based on the AIC was GTR + I + G for ITS, GTR + G for
tef1-
α
, and HKY + G for tub2. The ML analysis showed that of 2921 total characters, 2310
were constant, 475 were parsimony informative, and 136 were autapomorphic. The results
of both analyses showed that the isolates FM1-3, FM6 and 7, and FM9 were grouped in
the clade of B. dothidea (82/88, MP and ML bootstrap support %, respectively), the isolate
FA10 grouped within N. mediterraneum clade (97/97), and FA1-3, FM8, FB4, and FB6 were
grouped with the clade of N. parvum (97/98) (Figure 3). The conidia measurements were
(18.66)–22.7–(28.34)
×
(3.61)–4.9–(6.38) for B. dothidea, (14.0)–20.0–(27.2)
×
(4.3)–5.8–(6.8) for
N. mediterraneum, and (12.78)–15.1–(16.9) ×(4.16)–5.3–(7.21) for N. parvum.
Table 1.
Information on fungal isolates used in the phylogenetic analyses and their corresponding
GenBank accession numbers. Isolates in bold are from this study.
Scheme 1. Isolate ID ITS tef1-αtub2
Botryosphaeria agaves CBS 133992 = MFLUCC
11-0125T JX646791 JX646856 JX646841
B. agaves CBS 141505 = CPC 26299 KX306750 MT592030 MT592463
B. corticis CBS 119047T DQ299245 EU017539 EU673107
B. corticis CBS 119048 = CAP 198 DQ299246 EU017540 MT592464
B. dothidea CBS 115476 = CMW 8000T AY236949 AY236898 AY236927
B. dothidea CBS 110302 = CAP 007 AY259092 AY573218 EU673106
B. dothidea FM1 OM241975 OM262426 OM262439
B. dothidea FM2 OM241976 OM262427 OM262440
B. dothidea FM3 OM241977 OM262428 OM262441
B. dothidea FM6 OM241978 OM262429 OM262442
B. dothidea FM7 OM241979 OM262430 OM262443
B. dothidea FM9 OM241980 OM262431 OM262444
B. fabicerciana CBS 118831 = CMW 14009 DQ316084 MT592032 MT592468
B. fabicerciana CBS 127193 = CMW 27094T HQ332197 HQ332213 KF779068
B. kuwatsukai CGMCC 3.18007 KX197074 KX197094 KX197101
B. kuwatsukai CGMCC 3.18008 KX197075 KX197095 KX197102
B. qingyuanensis CERC 2946 = CGMCC
3.18742T KX278000 KX278105 KX278209
B. qingyuanensis CERC 2947 = CGMCC 3.18743 KX278001 KX278106 KX278210
B. ramosa CERC 2001 = CGMCC
3.187396 KX277989 KX278094 KX278198
B. ramosa CBS 122069 = CMW 26167T EU144055 EU144070 KF766132
Guignardia philoprina CBS 447.68 FJ824768 FJ824773 FJ824779
Neofusicoccum arbuti CBS 116131 = AR 4014T AY819720 KF531792 KF531793
N. arbuti CBS 117090 = UW13 AY819724 KF531791 KF531794
N. australe CBS 139662 = CMW 6837T AY339262 AY339270 AY339254
N. australe CMW 6853 AY339263 AY339271 AY339255
N. brasiliense CMM 1285 JX513628 JX513608 KC794030
N. brasiliense CMM 1338T JX513630 JX513610 KC794031
N. cordaticola CBS 123634 = CMW 13992T EU821898 EU821868 EU821838
N. cordaticola CBS 123635 EU821903 EU821873 EU821843
N. cryptoaustrale CBS 122813 = CMW 23785T FJ752742 FJ752713 FJ752756
N. dianense CSF6075 = CGMCC3.20082T MT028605 MT028771 MT028937
N. eucalypticola CBS 115679 = CMW 6539T AY615141 AY615133 AY615125
N. eucalypticola CBS 115766 = CMW 6217 AY615143 AY615135 AY615127
Plants 2022,11, 727 5 of 14
Table 1. Cont.
Scheme 1. Isolate ID ITS tef1-αtub2
N. eucalyptorum CBS 115791 = CMW 10125 =
BOT 24T AF283686 AY236891 AY236920
N. eucalyptorum CBS 145975 = CPC 29337 MT587477 MT592190 MT592682
N. hellenicum CERC 1947 = CFCC 50067T KP217053 KP217061 KP217069
N. hellenicum CERC 1948 = CFCC 50068 KP217054 KP217062 KP217070
N. hongkongense CERC2973 =
CGMCC3.18749T KX278052 KX278157 KX278261
N. hongkongense CERC 2968 = CGMCC 3.18748 KX278051 KX278156 KX278260
N. kwambonambiense CBS 123639 = CMW 14023T EU821900 EU821870 EU821840
N. kwambonambiense CBS 123641 = CMW 14140 EU821919 EU821889 EU821859
N. lumnitzerae CBS 139674 = CMW 41469T KP860881 KP860724 KP860801
N. lumnitzerae CBS 139675 = CMW 41228 MT587480 MT592193 MT592685
N. luteum CBS 110497 = CPC 4594 =
CAP 037 EU673311 EU673277 EU673092
N. luteum CBS 110299 = LM 926 = CAP
002T AY259091 KX464688 DQ458848
N. macroclavatum CBS 118223 = CMW 15955 =
WAC 12444T DQ093196 DQ093217 DQ093206
N. magniconidium CSF5876 = CGMCC3.20077T MT028612 MT028778 MT028944
N. mangiferae CBS 118531 = CMW 7024T AY615185 DQ093221 AY615173
N. mediterraneum CBS 121558 GU799463 GU799462 GU799461
N. mediterraneum CBS 121718 = CPC 13137T GU251176 GU251308 GU251836
N. mediterraneum FA10 OM241968 OM241976 OM262432
N. microconidium CERC3497 =
CGMCC3.18750T KX278053 KX278158 KX278262
N. microconidium CBS 118821 = CMW 13998 MT587497 MT592212 MT592704
N. ningerense CSF6028 = CGMCC3.20078T MT028613 MT028779 MT028945
N. nonquaesitum CBS 126655 = L3IE1 = PD484T GU251163 GU251295 GU251823
N. nonquaesitum CBS 133501 = UCR532 MT587498 MT592213 MT592705
N. occulatum CBS 128008 = MUCC 227T EU301030 EU339509 EU339472
N. occulatum MUCC 286 = WAC 12395 EU736947 EU339511 EU339474
N. parvum CBS 138823 = ICMP 8003 =
CMW 9081T AY236943 AY236888 AY236917
N. parvum CBS 110301 = CAP 074 AY259098 AY573221 EU673095
N. parvum FA1 OM241969 OM262420 OM262433
N. parvum FA2 OM241970 OM262421 OM262434
N. parvum FA3 OM241971 OM262422 OM262435
N. parvum FM8 OM241972 OM262423 OM262436
N. parvum FB4 OM241973 OM262424 OM262437
N. parvum FB6 OM241974 OM262425 OM262438
N. parviconidium CSF5667 = CGMCC3.20074T MT028615 MT028781 MT028947
N. pennatisporum WAC 13153 = MUCC 510T EF591925 EF591976 EF591959
N. pistaciae CBS 595.76T KX464163 KX464676 KX464953
N. podocarpi CBS 131677 = CMW 35494 MT587508 MT592223 MT592715
N. podocarpi CBS 131678 = CMW 35499 MT587509 MT592224 MT592716
N. protearum CBS 114176 = CPC 1775 = JT
189T AF452539 KX464720 KX465006
N. protearum CBS 115177 = CPC 4357 FJ150703 MT592239 MT592731
N. ribis CBS 115475 = CMW 7772T AY236935 AY236877 AY236906
N. ribis CBS 124923 = CMW 28320 FJ900608 FJ900654 FJ900635
N. ribis CBS 124924T FJ900607 FJ900653 FJ900634
N. ribis CBS 123645 = CMW 14058T EU821904 EU821874 EU821844
N. ribis CBS 123646 = CMW 14060 EU821905 EU821875 EU821845
N. sinense CGMCC3.18315T KY350148 KY817755 KY350154
N. sinoeucalypti CERC2005 =
CGMCC3.18752T KX278061 KX278166 KX278270
N. sinoeucalypti CERC3415 KX278063 KX278168 KX278272
N. stellenboschiana CBS 110864 = CPC 4598 AY343407 AY343348 KX465047
N. terminaliae CBS 125263 = CMW 26679T GQ471802 GQ471780 KX465052
N. terminaliae CBS 125264 = CMW 26683 GQ471804 GQ471782 KX465053
N. ursorum CBS 122811 = CMW 24480T FJ752746 FJ752709 KX465056
N. ursorum CBS 122812 = CMW 23790 FJ752745 FJ752708 KX465057
N. yunnanense CSF6142 = CGMCC3.20083T MT028667 MT028833 MT028999
N. viticlavatum CBS 112878 = CPC 5044 = JM
86T AY343381 AY343342 KX465058
N. viticlavatum CBS 112977 = STE-U 5041 AY343380 AY343341 KX465059
N. vitifusiforme CBS 110887 = CPC 5252 =
JM5T AY343383 AY343343 KX465061
N. vitifusiforme CBS 121112 = STE-U 5912 EF445349 EF445391 KX465016
Phyllosticta citricarpa CBS 102374 FJ824767 FJ538371 FJ824778
T: Type material.
Plants 2022,11, 727 6 of 14
Plants 2022, 11, x FOR PEER REVIEW 6 of 15
N. sinoeucalypti CERC3415 KX278063 KX278168 KX278272
N. stellenboschiana CBS 110864 = CPC 4598 AY343407 AY343348 KX465047
N. terminaliae CBS 125263 = CMW 26679T GQ471802 GQ471780 KX465052
N. terminaliae CBS 125264 = CMW 26683 GQ471804 GQ471782 KX465053
N. ursorum CBS 122811 = CMW 24480T FJ752746 FJ752709 KX465056
N. ursorum CBS 122812 = CMW 23790 FJ752745 FJ752708 KX465057
N. yunnanense CSF6142 = CGMCC3.20083T MT028667 MT028833 MT028999
N. viticlavatum CBS 112878 = CPC 5044 = JM 86T AY343381 AY343342 KX465058
N. viticlavatum CBS 112977 = STE-U 5041 AY343380 AY343341 KX465059
N. vitifusiforme CBS 110887 = CPC 5252 = JM5T AY343383 AY343343 KX465061
N. vitifusiforme CBS 121112 = STE-U 5912 EF445349 EF445391 KX465016
Phyllosticta citricarpa CBS 102374 FJ824767 FJ538371 FJ824778
T Type material.
Figure 3.
One of 100 equally parsimonious trees generated from maximum-parsimony analysis of the
three-gene (ITS + tef1-
α
+tub2) combined dataset from Botryosphaeriaceae species. Numbers in front
and after the slash represent parsimony and likelihood bootstrap values from 1000 replicates, respec-
tively. Isolates in red were generated in this study. Bar indicates the number of nucleotide changes.
2.3. Pathogenicity Test
The results of the pathogenicity test showed that all three species of Botryosphaeriaceae
identified in this study were pathogenic to F. microcarpa. Otherwise, the Eutypella sp.
isolate inoculated did not induce any lesions on the woody tissues, which was similar to
the control. For this reason, this species was excluded from the phylogenetic analyses.
External discoloration out of the inoculation point was observed after 7 days and all
the inoculated trees showed severe wood discoloration after the outer layer of bark was
removed (Figure 4A–D) Moreover, young twigs close to the inoculation point rapidly wilted
Plants 2022,11, 727 7 of 14
a few days after inoculation. Specifically, among the fungal species, the N. mediterraneum
isolate FA10 induced the longest lesions (mean 8.10 cm), followed by N. parvum isolate
FB4 (2.66 cm) and B. dothidea isolate FM2 (1.88 cm). All the inoculated species statistically
differed from the control (p< 0.05) (Figure 5). The colonies that emerged from the re-
isolations showed morphological characteristics (color, shape, and mycelium texture) that
fulfilled the Koch’s postulates.
Plants 2022, 11, x FOR PEER REVIEW 7 of 15
Figure 3. One of 100 equally parsimonious trees generated from maximum-parsimony analysis of
the three-gene (ITS + tef1-α + tub2) combined dataset from Botryosphaeriaceae species. Numbers in
front and after the slash represent parsimony and likelihood bootstrap values from 1000 replicates,
respectively. Isolates in red were generated in this study. Bar indicates the number of nucleotide
changes.
2.3. Pathogenicity Test
The results of the pathogenicity test showed that all three species of Botryosphaeriaceae
identified in this study were pathogenic to F. microcarpa. Otherwise, the Eutypella sp. iso-
late inoculated did not induce any lesions on the woody tissues, which was similar to the
control. For this reason, this species was excluded from the phylogenetic analyses. Exter-
nal discoloration out of the inoculation point was observed after 7 days and all the inocu-
lated trees showed severe wood discoloration after the outer layer of bark was removed
(Figure 4A–D) Moreover, young twigs close to the inoculation point rapidly wilted a few
days after inoculation. Specifically, among the fungal species, the N. mediterraneum isolate
FA10 induced the longest lesions (mean 8.10 cm), followed by N. parvum isolate FB4 (2.66
cm) and B. dothidea isolate FM2 (1.88 cm). All the inoculated species statistically differed
from the control (p < 0.05) (Figure 5). The colonies that emerged from the re-isolations
showed morphological characteristics (color, shape, and mycelium texture) that fulfilled
the Koch’s postulates.
Figure 4. Results of pathogenicity test after two weeks. (A) Neofusicoccum mediterraneum. (B) N.
parvum. (C) Botryosphaeria dothidea. (D) Control. Scale bar = 10 cm.
Figure 4.
Results of pathogenicity test after two weeks. (
A
)Neofusicoccum mediterraneum. (
B
)
N. parvum. (C)Botryosphaeria dothidea. (D) Control. Scale bar = 10 cm.
Plants 2022, 11, x FOR PEER REVIEW 8 of 15
Figure 5. Comparisons of average lesion length (cm) resulting from pathogenicity test among B.
dothidea, N. mediterraneum and N. parvum on potted plants. Columns are the means of 15 inoculation
points (five per plant) for each fungal species. Control consisted of 12 inoculation points. Vertical
bars represent the standard error of the means. Bars topped with different letters indicate treatments
that were significantly different according to Fisher’s protected LSD test (α = 0.05).
3. Discussion
The results of our study confirm, for the first time, the presence of three species, B.
dothidea, N. mediterraneum, and N. parvum, affecting F. microcarpa in Italy. Regarding Bot-
ryosphaeriaceae, little is known about its association with F. microcarpa. According to the
U.S. National Fungus Collections Fungal Database [9], only a few, old reports describe the
association of Lasiodiplodia theobromae (as Botryodiplodia theobromae) in Pakistan [31] and
Egypt [29], and Diplodia fici-retusae in Taiwa n on Ficus retusa (synonymous of F. microcarpa)
[32,33]. A commonly reported disease of F. microcarpa, as well as other Ficus spp., is “sooty
canker”, which is caused by Neoscytalidium dimidiatum (traditionally reported also as Hen-
dersonula toruloidea and Natrassia mangiferae). The pathogen, as well as other Botryosphaeri-
aceae, induces cankers and dieback, often accompanied by a powdery mass of black spores
(arthroconidia) produced by this species [14,18–21,26,28,34]. Recently, in California B.
dothidea, N. luteum, N. mediterraneum, and N. parvum were reported as causing branch can-
kers and dieback on F. microcarpa trees in Los Angeles County [25]. In recent years, Botry-
osphaeriaceae spp. have been reported attacking many different crops in Italy, and, espe-
cially in Sicily, it is well known that these species spread from nurseries to the open field,
from ornamental plants to the agricultural ones. Specifically, B. dothidea has recently been
reported in Sicily on walnut and pistachio [35,36]. Moreover, N. mediterraneum and N. par-
vum have been reported as highly aggressive pathogens among the Botryosphaeriaceae in
Sicily [13,35,37,38]. In addition, N. mediterraneum was the most encountered species in Si-
cilian pistachio orchards [36]. From this and previous studies conducted in Sicily, it
emerged that Botryosphaeriaceae spp., and especially the species described in this study,
are easily encountered in different hosts and landscapes. Regarding the ecology of these
fungi, it is well known that they are also endophytes on many hosts [11], often coexisting
in the same tissues [39] and forming long latent infections [40,41]. This must be taken into
serious consideration, since many infections can spread from nurseries (as latent infec-
tions) to open fields. Recently, studies conducted in California on latent infections on nut
crops helped us to properly quantify these pathogens using real-time PCR assays
[40,42,43]. The ability of these fungi to disperse their spores (conidia) by wind, rain, and
insects [10] in conjunction with intercontinental human movements with no adequate
quarantine strategies led them to easily spread all over the world [44], as demonstrated
Figure 5.
Comparisons of average lesion length (cm) resulting from pathogenicity test among
B. dothidea
,N. mediterraneum and N. parvum on potted plants. Columns are the means of 15 inoculation
points (five per plant) for each fungal species. Control consisted of 12 inoculation points. Vertical
bars represent the standard error of the means. Bars topped with different letters indicate treatments
that were significantly different according to Fisher’s protected LSD test (α= 0.05).
3. Discussion
The results of our study confirm, for the first time, the presence of three species,
B. dothidea,N. mediterraneum, and N. parvum, affecting F. microcarpa in Italy. Regarding
Botryosphaeriaceae, little is known about its association with F. microcarpa. According to the
Plants 2022,11, 727 8 of 14
U.S. National Fungus Collections Fungal Database [
9
], only a few, old reports describe
the association of Lasiodiplodia theobromae (as Botryodiplodia theobromae) in Pakistan [
31
]
and Egypt [
29
], and Diplodia fici-retusae in Taiwan on Ficus retusa (synonymous of F. micro-
carpa) [
32
,
33
]. A commonly reported disease of F. microcarpa, as well as other Ficus spp.,
is “sooty canker”, which is caused by Neoscytalidium dimidiatum (traditionally reported
also as Hendersonula toruloidea and Natrassia mangiferae). The pathogen, as well as other
Botryosphaeriaceae, induces cankers and dieback, often accompanied by a powdery mass
of black spores (arthroconidia) produced by this species [
14
,
18
–
21
,
26
,
28
,
34
]. Recently, in
California B. dothidea,N. luteum,N. mediterraneum, and N. parvum were reported as causing
branch cankers and dieback on F. microcarpa trees in Los Angeles County [
25
]. In recent
years, Botryosphaeriaceae spp. have been reported attacking many different crops in Italy,
and, especially in Sicily, it is well known that these species spread from nurseries to the
open field, from ornamental plants to the agricultural ones. Specifically, B. dothidea has
recently been reported in Sicily on walnut and pistachio [
35
,
36
]. Moreover, N. mediter-
raneum and N. parvum have been reported as highly aggressive pathogens among the
Botryosphaeriaceae in Sicily [
13
,
35
,
37
,
38
]. In addition, N. mediterraneum was the most encoun-
tered species in Sicilian pistachio orchards [
36
]. From this and previous studies conducted
in Sicily, it emerged that Botryosphaeriaceae spp., and especially the species described in
this study, are easily encountered in different hosts and landscapes. Regarding the ecology
of these fungi, it is well known that they are also endophytes on many hosts [
11
], often
coexisting in the same tissues [
39
] and forming long latent infections [
40
,
41
]. This must
be taken into serious consideration, since many infections can spread from nurseries (as
latent infections) to open fields. Recently, studies conducted in California on latent infec-
tions on nut crops helped us to properly quantify these pathogens using real-time PCR
assays [
40
,
42
,
43
]. The ability of these fungi to disperse their spores (conidia) by wind, rain,
and insects [
10
] in conjunction with intercontinental human movements with no adequate
quarantine strategies led them to easily spread all over the world [
44
], as demonstrated
for N. parvum, the most adapted organism, which is detected from the north to the south,
excluding boreal forests and montane grasslands [
45
]. Many factors can be involved in
the ability of some Botryosphaeriaceae species to jump from one host to another, meaning
that they are more virulent than other species. Among these, a recent study [
46
] revealed
how some groups of taxa, such as Botryosphaeria,Lasiodiplodia, and Neofusicoccum, show an
expansion of certain clades of gene families involved in the pathogenesis. Specifically, in
the Botryosphaeria and Neofusicoccum genomes, an expansion of secreted cell-wall-degrading
enzymes (CAZymes) was observed [
46
]. It is no surprise that the species identified in
this study also occurred on other taxonomically distant hosts in Sicily. Batista et al. [
45
]
showed that B. dothidea is associated with 403 hosts in 66 countries, and N. parvum with
223 hosts in 50 countries. In recent decades, in Sicily, a relevant increase was observed
in Botryosphaeriaceae in nurseries, as well as in open fields (Polizzi G., unpublished data).
Botryosphaeriaceae disease expression is strongly related to stresses due to factors other
than the Botryosphaeriaceae infection itself [
47
–
49
]. Related to this, it should be noticed
that climate change contributes to additional stress or pressure on woody plants through
extreme weather conditions or the expansion of pathogens’ host ranges [
11
]. In fact, climate
change affects the dynamics of fungal populations, in terms of biology and ecology [
49
].
Gange et al. [
50
] conducted a study in the UK on the species Auricularia auricula-judae,
demonstrating an alteration in the phenology (the earlier appearance of fruit bodies and
a longer fruiting period) and an expansion of the host range consistent with a response
to observed warming trends in the climate, also suggesting that climate change affected
the interactions between wood-inhabiting fungi. Combative interactions are considered
the main drivers of fungal community development in decaying wood [
51
,
52
], and these
can be strongly affected by temperature, water potential, gaseous regime, and resource
size [
53
–
55
]. All these factors contribute to making Botryosphaeriaceae disease severe and
ubiquitous, compared with otherwise “mild diseases” [
56
]. Urban areas, which are even
less investigated than agricultural ones, must be considered crucial routes of introduction
Plants 2022,11, 727 9 of 14
and dissemination for Botryosphaeriaceae [
57
]. It is well known that stressed trees are much
more predisposed to Botryosphaeriaceae disease [
11
,
58
], and this should be taken into careful
consideration regarding ornamental trees in urban landscapes. In fact, trees grown in urban
areas can also be considered more exposed to stress factors [
59
], and thus more susceptible
to Botryosphaeriaceae disease. This could represent a serious threat in urban areas, not only
in terms of aesthetic damage, but mostly in terms of public safety. In relation to these
predisposing factors, we ascertained during our investigation that F. microcarpa trees grown
in the urban areas of Catania and Siracusa provinces were severely and improperly pruned,
especially during the humid seasons. In order to avoid the spread of Botryosphaeriaceae
species, some recommendations should be taken into serious consideration. Since it is
known that both rainfall and fog [
60
,
61
] positively affect the release of Botryosphaeriaceae
spores, farmers or pruning crews should not prune when rain is forecasted or with dense
fog to avoid the contamination of fresh wounds by Botryosphaeriaceae [
62
]. Moreover,
recommendations as to pruning type depend on the tree species, which is why trained
pruning crews should be selected for this crucial practice. As demonstrated on pistachio,
Botryosphaeria panicle and shoot blight were reduced by 50–60% by trained pruning crews
compared to the disease levels in trees pruned by unspecialized crews [
63
]. Furthermore,
in California, field experiments conducted on F. carica affected by fig limb dieback demon-
strated that pruning 5 cm below the canker successfully removed the pathogen from the
tissues [
36
]. Regarding trained pruning crews, it is crucial that workers disinfect their
pruning tools, since these could easily transmit inoculum (spores, mycelium, and fruit
bodies) from one tree to another. As demonstrated on walnut, pathogen spores were trans-
ferred from the chainsaws to the agar media, whereas Botryosphaeriaceae species were not
found when the chainsaws were disinfected with a 2% dilution of vinegar or commercial
household bleach (T.J. Michailides, unpublished data/personal communication). In addi-
tion, the usage of biological control agents as protectants for pruning wounds, especially
in urban areas, should be considered. Encouraging results have been obtained on other
crops, such as almond and grapevine treated with Trichoderma-based formulants against
canker pathogens [
64
–
66
]. Further investigations need to be conducted in this direction.
Good agronomic practices and, possibly, the usage of biocontrol agents, can help us to
control Botryosphaeriaceae disease in urban areas. To our knowledge, this is the first study of
Botryosphaeriaceae disease on F. microcarpa in Europe.
4. Materials and Methods
4.1. Surveys and Fungal Isolations
During the years between 2019 and 2020, surveys were carried out in numerous
urban areas of the cities of Catania (Catania province), and Siracusa (Siracusa province),
Sicily, where F. microcarpa were the most prevalent ornamental trees, including tree-lined
streets, gardens, public parks, and squares. Several symptomatic samples obtained from
ten plants were collected and brought to the laboratory of the Dipartimento di Agricoltura,
Alimentazione e Ambiente, University of Catania, for further investigations. For culture
isolation, small sections (0.2 to 0.3 cm
2
) of symptomatic tissues (branches and shoots) were
surface-disinfected for 1 min in 1.5% sodium hypochlorite, rinsed in sterile water, dried
on sterile absorbent paper under laminar hood and placed on potato dextrose agar (PDA,
Lickson, Vicari, Italy) amended with 100 mg/liter of streptomycin sulfate (Sigma-Aldrich,
St. Louis, MO, USA) (PDAS) to prevent bacterial growth, and then incubated at 25
±
1
◦
C
for 3–5 days until fungal colonies were large enough to be examined. Subsequently, colonies
of interest were transferred to fresh PDAS to make pure cultures, and then single-hyphal
tip cultures were obtained and maintained on PDAS at 25
±
1
◦
C. Isolates characterized
in this study were stored in the fungal collection of the laboratory with the labels FA, FB,
and FM.
Plants 2022,11, 727 10 of 14
4.2. Morphological and Molecular Characterization
For the morphological characterization of the pathogens, the length and width of
50 conidia from the 21-day-old colonies of the isolates FM1, FA10, and FA1 grown on
PDA were measured using a fluorescence microscope (Olympus-BX61) coupled to an
Olympus DP70 digital camera; measurements were captured using software analysis
3.2 (Soft Imaging System GmbH, Münster, Germany). Dimensions are reported as the
minimum and maximum in parentheses and the average. Total fungal DNA was extracted
using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI,
USA), scraping the mycelium with a sterile scalpel from 5-day-old fungal cultures grown
on PDA or malt extract agar (MEA, Oxoid LTD. Basingstoke, Hampshire, England) media.
The genomic DNA extracted was visualized on 1% agarose gels (90 V for 40 min) stained
with GelRed
®
(Biotium, Fremont, CA, USA). The quality of the DNA was determined
through Nanodrop Lite Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE,
USA). The internal transcriber spacer region (ITS) of the nuclear ribosomal RNA operon
was amplified with primers ITS5 (5
0
-GGA AGT AAA AGT CGT AAC AAG G-3
0
) and ITS4
(5
0
-TCC TCC GCT TAT TGA TAT GC-3
0
) [
67
]; the primers EF1-728F (5
0
-CAT CGA GAA
GTT CGA GAA GG-3
0
) and EF1-986R (5
0
-TAC TTG AAG GAA CCC TTA CC-3
0
) [
68
] were
used to amplify part of the translation elongation factor 1alpha gene (tef1-
α
); and primer
sets Bt2a (5
0
-GGT AAC CAA ATC GGT GCT TTC-3
0
) and Bt2b (5
0
-ACC CTC AGT GTA
GTG ACC CTT GGC-3
0
) [
69
] were used for the partial beta tubulin (tub2). Amplification
by polymerase chain reaction (PCR) was performed in a total volume of 25
µ
L using
One Taq
®
2X Master Mix with Standard Buffer (BioLabs, New England, NEB), according
to the manufacturer’s instructions, on an Eppendorf Mastercycler (AG 22331 Hamburg,
Germany). The thermal cycle consisted of initial 30 s at 94
◦
C, followed by 35 cycles at 94
◦
C
for 30 s, 49
◦
C (ITS), 57–59
◦
C (tef1-
α
), or 52
◦
C (tub2) for 1 min, 68
◦
C for 1 min, and 5 min
at 68 ◦C. Regarding Botryosphaeriaceae, in total, 45 isolates were sequenced (tub2) and only
13 representative isolates were considered for further gene sequencing and phylogenetic
analyses. Concerning the Eutypella-like species, a total of 7 isolates were sequenced (ITS
and tub2). PCR products were visualized on 1% agarose gels (90 V for 40 min), purified, and
sequenced by Macrogen Inc. (Seoul, South Korea). Forward and reverse DNA sequences
were assembled and edited using MEGA X: Molecular Evolutionary Genetics Analysis
across computing platforms [70] and submitted to GenBank.
4.3. Phylogenetic Analysis
Chromatograms were viewed using FinchTV Version 1.4.0 (Geospiza, Inc.; Seattle,
WA, USA; http://www.geospiza.com (accessed on 16 February 2022)). Sequences were
read and edited using MEGAX. Before constructing the phylogenetic tree, BLAST searches
were performed using the NCBI nucleotide database [
71
]. ITS, tef1-
α
, and tub2 DNA
sequence datasets were aligned using MEGAX, and manual alignments were performed
when necessary. A partition-homogeneity test with heuristic search and 1000 homogeneity
replicates was performed using PAUP* (Phylogenetic Analysis Using Parsimony) version
4.0a (Sinauer Associates, Sunderland, MA, USA) [
72
] to test for discrepancies in the three-
gene dataset. For comparison, 79 additional sequences were selected according to the
recent literature on the Botryosphaeriaceae [
73
,
74
] to be included in the alignment (Table 1).
Maximum parsimony analysis (MP) was performed in PAUP v.4.0a. The analysis of the
combined dataset (ITS + tef1-
α
+tub2) was performed with the heuristic search function and
tree bisection and reconstruction (TBR) as branch-swapping algorithms with the branch-
swapping option set to ‘best trees’ only. Gaps were treated as ‘missing’, the characters
were unordered and of equal weight, and Maxtrees were limited to 100. Tree length (TL),
consistency index (CI), retention index (RI), and rescaled consistency index (RC) were
calculated. To identify the best-fit model of nucleotide evolution for each gene according to
the Akaike information criterion (AIC), MrModeltest v. 2.4 [
75
] was used. The maximum
likelihood analysis (ML) of the combined genes was performed in GARLI v.0.951 [
76
].
For both analyses, clade support was assessed by 1000 bootstrap replicates. Guignardia
Plants 2022,11, 727 11 of 14
philoprina (CBS 447.68) and Phyllosticta citricarpa (CBS 102374) served as the outgroup in
both analyses.
4.4. Pathogenicity Test
Pathogenicity tests were conducted on potted, healthy, 4-year-old F. microcarpa plants
maintained at room temperature. For each fungal species, one representative isolate was
inoculated. Specifically, three plants were used for each isolate, and five inoculation points
were chosen along the trunk on each plant (~30 cm distant one from each other). The
inoculation site was first surface-disinfected by spraying with 70% ethanol solution, and
wounds were made with a sterilized 6-millimeter cork borer after removing the bark, and a
mycelium plug (6 mm in diameter) was placed upside down into the plant tissue wound.
Wounds were sealed with Parafilm
®
(Pechney Plastic Packaging Inc., Chicago, IL, USA).
In total, 12 additional wounds were inoculated with sterile PDA plugs as controls. Plants
were regularly watered. The presence and length of the resulting lesions were recorded
two weeks after the inoculation. Lesion length measurements were analyzed in Statistix
10 [
77
] via analysis of variance (ANOVA), and mean differences were compared with the
Fisher’s protected least significant difference (LSD) test at
α
= 0.05. In order to fulfil Koch’s
postulates, re-isolations were carried out on PDAS following the procedure described above.
Each re-isolated fungus was identified through the observation of colony characteristics.
5. Conclusions
In the present study, three species of Botryosphaeriaceae were isolated from symptomatic
samples of F. microcarpa showing severe symptoms of cankers, wood discolourations,
bark cracking, and dieback. Morphological and molecular tools identified B. dothidea,
N. mediterraneum
, and N. parvum. Pathogenicity tests fulfilled Koch’s postulates. The
results of this study provide new information on this important family of phytopathogenic
fungi and its wide host range. This is the first report in Europe of Botryosphaeriaceae affecting
F. microcarpa.
Author Contributions:
Conceptualization, D.A., A.F. and G.P.; methodology, A.F., M.B.C. and G.G.;
software, G.G.; validation, G.P.; formal analysis, G.G.; investigation, G.P.; resources, G.P.; data
curation, G.G.; writing—original draft preparation, G.G.; writing—review and editing, D.A., A.F.,
G.G. and G.P.; visualization, G.P.; supervision, D.A. and G.P.; project administration, G.P.; funding
acquisition, G.P. All authors have read and agreed to the published version of the manuscript.
Funding:
Programma Ricerca di Ateneo MEDIT-ECO UNICT 2020–2022 Linea 2-University of Catania
(Italy); Starting Grant 2020, University of Catania (Italy); Fondi di Ateneo 2020–2022, University of
Catania (Italy), Linea Open Access. Research Project 201–62018, University of Catania 5A722192134.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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