<em>Coniella granati</em> e <em>Phytophthora palmivora</em>: i principali patogeni coinvolti nell’eziologia dei fenomeni di disseccamento e moria del melograno nell’Italia nord-orientale
10.6092/issn.2531-7342/11170
Nel corso degli ultimi anni sono stati osservati gravi fenomeni di deperimento e morie di piante di melograno in vari impianti del Veneto. Vista l’assenza di informazioni sull’eziologia di queste fitopatie, è stata svolta un’indagine diagnostica con l’intento di isolare, identificare e caratterizzare i patogeni coinvolti. Dagli isolamenti effettuati su campioni di branche e rizosfera prelevati da 45 piante sintomatiche sono state ottenute complessivamente 88 colonie di specie fungine e di oomiceti, che sulla base dei caratteri morfologici e genetici (sequenze nucleotidiche dell’intera regione degli spaziatori interni trascritti ITS1 e ITS2) sono state suddivise in 8 gruppi appartenenti rispettivamente alle specie: Botryosphaeria dothidea e Neofusicoccum parvum (Botryosphaeriaceae), Diaporthe eres (Diaporthaceae), Coniella granati (Schizoparmaceae), Cytospora punicae (Valsaceae), Phytophthrora palmivora, P. pseudocryptogea e Phytopythium vexans (Peronosporaceae). Nei saggi di patogenicità, tutte le specie, ad eccezione di C. punicae, hanno riprodotto i sintomi della malattia osservati in campo, sebbene con un diverso grado di virulenza. I risultati ottenuti evidenziano la complessità eziologica dei fenomeni di disseccamento e moria di piante osservati nei melograneti dell’Italia nord-orientale. Ulteriori studi sono attualmente in corso per determinare eventuali interazioni sinergiche tra i patogeni riscontrati sulla chioma delle piante di melograno e quelli ad habitus radicale
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<em>Coniella granati</em> and <em>Phytophthora palmivora</em> the main pathogens involved in pomegranate dieback and mortality in north-eastern Italy
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Xavier KV, Nepal Kc A, Crous PW, Groenewald JZ, Vallad GE (2019) Dwiroopa punicae sp. nov. (Dwiroopaceae fam. nov., Diaporthales), associated with leaf spot and fruit rot of pomegranate (Punica granatum). Fungal Systematics and Evolution 4:33–41. https://doi.org/10.3114/fuse.2019.04.04
<em>Coniella granati</em> and <em>Phytophthora palmivora</em> the main pathogens involved in pomegranate dieback and mortality in north-eastern Italy
https://italianmycology.unibo.it/article/download/11170/11504
49
Linaldeddu et al.                                                           Italian Journal of Mycology 49: 92-100 (2020)

DOI: 10.6092/issn.2531-7342/11170

Original paper

 Coniella granati and Phytophthora palmivora: the main pathogens
          involved in pomegranate dieback and mortality
                       in north-eastern Italy
               Benedetto T. Linaldeddu, Carlo Bregant, Barbara Ruzzon, Lucio Montecchio

1 Dipartimento Territorio e Sistemi Agro-Forestali, Università di Padova, Viale dell’Università 16, 35020 Legnaro (PD),
Italy

Corresponding author e-mail: benedetto.linaldeddu@unipd.it


Article info
Received 15/06/2020; accepted 06/08/2020
DOI: 10.6092/issn.2531-7342/11170

Abstract
Severe branch dieback, root rot and sudden death symptoms have recently been observed on pomegranate in several
orchards in Veneto (north-eastern Italy). Since there is little information about the aetiology of these diseases and given
the growing economic interest in this crop, an in-depth study was conducted. From winter 2017 to autumn 2018, forty-
five symptomatic plants in two orchards were monitored and sampled to isolate the associated pathogens. Based on
morphology, colony appearance and DNA sequence data, eight species belonging to seven genera and five families
were isolated and identified. These included Botryosphaeria dothidea and Neofusicoccum parvum (Botryosphaeriaceae),
Diaporthe eres (Diaporthaceae), Coniella granati (Schizoparmaceae), Cytospora punicae (Valsaceae), Phytophthrora
palmivora, Phytophthora pseudocryptogea and Phytopythium vexans (Peronosporaceae). Pathogenicity trials conducted
on 3-year-old pomegranate seedlings showed that three species, C. granati, N. parvum and P. palmivora are aggressive
pathogens on pomegranate. Results obtained have allowed us to establish that the number of pathogens associated with
emerging diseases of pomegranate is greater than previously recognised. The species B. dothidea, P. pseudocryptogea and
P. vexans are reported for the first time on pomegranate in Italy.

Keywords
emerging diseases; Neofusicoccum; Botryosphaeria; Phytopythium; Cytospora; pathogenicity


Introduction

Pomegranate (Punica granatum L.) is a small shrub native to the region extending from Iran to
northern India (Mars, 2000). It has been cultivated for its edible fruits throughout the Mediterranean
region since ancient times (Adiletta et al., 2018). Its adaptation to the Mediterranean climate has
contributed to a wide diffusion in various countries, originating several local genotypes varying
from sweet to sour in fruit taste and morphology (Holland et al., 2008; Ferrara et al., 2011). In Italy
pomegranate cultivation is concentrated chiefly in the southern regions where several local genotypes
are present (Calani et al., 2013; Ferrara et al., 2014).
The growing consumption of pomegranate fruits for their beneficial nutritional and medicinal properties
in preventing cancer and cardiovascular disease, has favoured a rapid expansion of cultivation of this
crop in several countries outside the traditional areas (Lansky and Newman, 2007; Seeram et al.,
2008; Basu and Penugonda, 2009).
Together with the expansion of cultivation areas, there has been an increase of new disease reports
for this crop worldwide, including foliar and fruit diseases caused by Colletotrichum gloeosporioides

ISSN 2531-7342 Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2020 The Authors. This work is licensed under CC BY 4.0
Linaldeddu et al.                                                       Italian Journal of Mycology 49: 92-100 (2020)

DOI: 10.6092/issn.2531-7342/11170


(Penz.) Penz. & Sacc. and Dwiroopa punicae K.V. Xavier, A.N. KC, J.Z. Groenew., Vallad & Crous
(Uysal and Kurt, 2019; Xavier et al., 2019), branch cankers and dieback caused by Cytospora punicae
Sacc., Coniella granati (Sacc.) Petr. & Syd. and Neofusicoccum parvum (Pennycook & Samuels)
Crous, Slippers & A.J.L. Phillips (KC and Vallad, 2016; Thomidis and Exadaktylou, 2011; Triki et al.,
2015) and root and collar rot caused by Phytophthora spp. (Kurbetli et al., 2020). In particular, in Italy
severe attacks of N. parvum and C. granati have recently been reported in the main growing areas
of central and southern regions (Pollastro et al., 2016; Riccioni et al., 2017). Since 2017, a serious
decline of pomegranate plants with a notable impact on their vitality and productivity has also been
observed in several orchards in Veneto (north-eastern Italy).
Since there is no information about the aetiology of this emerging disease, a survey was conducted to
establish the causal agents.

Materials and Methods
Field surveys and sampling procedure
From winter 2017 to autumn 2018, the health status of pomegranate plants was monitored in two
orchards located in Veneto (north-eastern Italy) (Table 1). The plants were checked for the presence of
symptoms on branches and trunk (dieback, exudates, cankers and epicormic shoots), and in the collar
and root system (exudates, necrosis and loss of fine roots).
A total of 40 symptomatic plants were randomly chosen for sampling. From each plant a cankered
branch and a portion of soil (300 g) including fine roots around the collar was sampled. Additional
necrotic root samples were collected from five plants showing sudden death symptoms in the site 2
(Table 1).

     Table 1 - Information of sampling sites and number of symptomatic plants sampled

       Study            Geographic                                                           Number of
        sites                                                Cultivar
                        coordinates                                                       plant monitored
          1       45°36’58”N - 12°28’15”E               Mollar de Elche                          10
          2       45°56’20”N - 12°18’14”E               Dente di Cavallo                        35*
    *Including five plants with sudden death symptoms


Isolation of canker-causing agents
All branch samples were taken to the laboratory to be visually inspected and the outer bark surface
was removed with a sterile scalpel. Longitudinal and transversal cuts were made to observe any
internal symptom. Isolations were made from approx. 5 mm2 chips of inner bark and xylem cut
aseptically from the margin of necrotic lesions (Smahi et al., 2017). All chips were placed on 90 mm
Petri dishes containing potato dextrose agar (PDA, Oxoid Ltd). After incubation at 25 ± 1 °C for 5–7
days in the dark, hyphal tips from the emerging fungal colonies were sub-cultured onto half-strength
PDA and incubated at room temperature under natural daylight to enhance sporulation.

Isolation of root rot pathogens
In the laboratory rhizosphere soil samples were placed in a plastic box and flooded with 2 L of distilled
water. After 24 h, young cork oak and elder leaves were placed on the water surface and used as baits.
Boxes were kept at 18-20 °C under natural daylight and after 3-5 days, leaves showing dark spots
were cut in pieces of 5 mm2 and placed on 90 mm Petri dishes containing PDA supplemented with
100 ml/L of carrot juice, 0.015 g/L of pimaricin and 0.05 g L-1 of hymexazol (PDA+) (Linaldeddu et
al., 2020).
Isolation of Phytophthora was also directly attempted from roots of five plants showing sudden death


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symptoms. Necrotic roots were cut in 3 cm long samples, rinsed in distilled water, blotted dry on filter
paper and then small inner bark pieces were cut from the margin of necrotic lesions and placed onto
PDA+.
Petri dishes were incubated in the dark at 20 °C and examined every 12 h. Hyphal tips from the
emerging colonies were sub-cultured on carrot agar (CA) (Erwin and Ribeiro, 1996) and PDA and
incubated at 20 °C in the dark. To enhance sporangia production, CA plugs (5 mm diameter) of
each isolate were placed in Petri dishes containing unsterile pond water. Sporangial production was
assessed every 12 h for 4 days by microscopic observation.

Identification of isolates
All isolates were initially grouped in morphotypes on the basis of colony growth characteristics
including surface and reverse colony appearance observed after 7 days of incubation on PDA and CA
at 25 °C in the dark and morpho-biometric data of conidia or sporangia. Measurements of conidia
and sporangia were taken with the software Motic Images Plus 3.0 paired with a Moticam 10+
camera connected to a Motic BA410E microscope. In particular, Phytophthora isolates were grouped
according to morphological descriptions provided by Erwin and Ribeiro (1996).
Molecular analysis was used to confirm the identity of all isolates at species level. InstaGene Matrix
(BioRad Laboratories, Hercules) was used to extract genomic DNA from mycelium of 5-day-old
cultures grown on PDA and incubated at 25 °C in the dark. The primers ITS1 and ITS4 (White et al.,
1990) were used to amplify and sequence the internal transcribed spacer (ITS) regions, including the
complete 5.8S gene. Polymerase chain reaction (PCR) mixtures and amplification conditions were
as described by Linaldeddu et al. (2016). The PCR products were purified using a EUROGOLD gel
extraction kit (EuroClone S.p.A.) following the manufacturer’s instructions.
The ITS regions were sequenced by the BMR Genomics DNA sequencing service (www.bmr-genomics.
it), in both directions, with the primers used for amplification. The nucleotide sequences were read
and edited with FinchTV 1.4.0 (Geospiza, Inc. http://www.geospiza.com/finchtv) and then compared
with reference sequences (type material) retrieved from GenBank using the BLASTn algorithm. ITS
sequences from representative isolates obtained in this study were deposited in GenBank (www.ncbi.
nlm.nih.gov/genbank).

Pathogenicity test
The pathogenicity of 7 isolates (4 fungi and 3 oomycetes), representative of the seven main species
isolated in this study, was assessed on 3-year-old pomegranate seedlings in March-June 2019. Ten
seedlings were inoculated with each isolate, and ten seedlings were used as controls.
The fungal isolates were inoculated in the main stem. The inoculation point was initially surface-
disinfected with 90% ethanol and then a small U-shaped wound made with a flamed scalpel was
inoculated with an agar-mycelium plug taken from the margin of an actively growing colony on PDA.
The inoculation site was covered with cotton wool soaked in sterile water and wrapped in a piece of
aluminium foil secured with masking tape. Controls were inoculated with a sterile PDA plug applied
as described above.
The oomycetes were inoculated at the collar. After disinfecting the bark with 90% ethanol, the
seedlings were inoculated under the bark after removing a piece of outer bark (5 mm Ø) with a sterile
cork borer. A same-sized agar-mycelium plug cut from the margin of a 4-day-old CA colony was
used as inoculum (Linaldeddu et al., 2020). The inoculation point was protected as described above.
Ten control plants were inoculated with a sterile CA plug. All inoculated plants were kept in field
conditions at 6.1 to 37.6 °C and watered regularly for 3 months.
Re-isolation of inoculated species was attempted by transferring 10 pieces of inner bark and wood
tissues taken around the margin of each lesion onto PDA (fungi) and PDA+ (oomycetes). Cultures


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obtained were grown in daylight at room temperature and then identified by morphological and
molecular analysis (ITS region).

Statistical analyses
Pathogenicity assay data were checked for normality, then subjected to analysis of variance (ANOVA).
Significant differences among mean values were determined using Fisher’s least significant differences
(LSD) multiple range test (P = 0.05) using XLSTAT software (Addinsoft).

Results

Field surveys
Field inspections conducted over a two-year period showed the widespread presence of symptomatic
pomegranate plants in both orchards. Disease incidence (estimated as number of symptomatic plants
out of the total number of plants) ranged from 54% (site 2) to 71% (site 1) and the rate of mortality
from 17% to 43%. Plants showed a complex symptomology including leaf chlorosis, shoot blight,
branch canker and dieback, and epicormic shoots on trunk (Fig. 1a, b); in addition, several plants
displayed sudden death symptoms (Fig. 1c).




  Fig. 1 - Main disease symptoms detected on pomegranate plants: progressive branch dieback and leaf chlorosis
  (a, b); sudden death (c); dead plant (d). Colony morphology of Botryosphaeria dothidea (e), Coniella granati (f),
  Cytospora punicae (g), Diaporthe eres (h), Neofusicoccum parvum (i), Phytophthora palmivora (j), Phytophthora
  pseudocryptogea (k) and Phytopythium vexans (l) after 7 days growth at 25 °C on PDA in the dark. Symptoms
  observed on pomegranate seedlings 90 days after inoculation with: B. dothidea (m), C. granati (n), C. punicae (o,
  p), N. parvum (q), P. palmivora (r), P. pseudocryptogea (s) and P. vexans (t). Asymptomatic control seedlings (u, v).

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Aetiology
Isolations performed from 40 cankered branch samples yielded a total of 51 fungal colonies. On the
basis of morphological features and DNA sequence data (ITS region), five distinct species namely
C. granati (26 isolates from both sites), Botryosphaeria dothidea (Moug.) Ces. & De Not. (9 isolates
from both sites), C. punicae (8 isolates from both sites), N. parvum (6 isolates from the site 1) and
Diaporthe eres Nitschke (2 isolates from the site 1) were identified (Fig. 1e-l).
Out of 40 plants investigated 36 were found to be infected by Phytophthora species. In total, 37
isolates belonging to two species, namely Phytophthora palmivora (E.J. Butler) E.J. Butler and P.
pseudocryptogea Safaief., Mostowf., G.E. Hardy & T.I. Burgess were obtained from rhizosphere
soil samples collected around the plant collar. All isolates conformed morphologically to the formal
description of each Phytophthora species available in the literature (Erwin and Ribeiro, 1996;
Safaiefarahani et al., 2015).
Phytophthora palmivora was the most common species, with an isolation frequency of 72.5%, whereas
P. pseudocryptogea was isolated from the 20% of plants. Furthermore, P. palmivora was the only
species obtained from the necrotic roots collected from the five plants with sudden death symptoms
(Fig. 1c). From rhizosphere soil samples of 12 plants of the site 2, colonies of Phytopythium vexans
(de Bary) Abad, de Cock, Bala, Robideau, A.M. Lodhi & Lévesque were also obtained.
For each species BLAST searches against GenBank showed 100% identity to reference sequences
of ex-type isolates and/or representative strains. The ITS sequence of a representative isolate of
each species was deposited in GenBank: B. dothidea (MT611222), C. granati (MT611223), C.
punicae (MT611224), D. eres (MT611225), N. parvum (MT611226), P. palmivora (MT611227), P.
pseudocryptogea (MT611228) and P. vexans (MT611229).

Pathogenicity
At the end of the experimental period, all seedlings inoculated with B. dothidea, C. granati, N.
parvum, P. palmivora, P. pseudocryptogea and P. vexans displayed necrotic lesions that spread up and
down from the inoculation site (Fig. 1), whereas no disease symptoms were observed on seedlings
inoculated with C. punicae and the stem wounds had begun to heal (Fig. 1o, p).
The average lesion length caused by the four fungal species differed significantly (Fig. 2a). Lesions
caused by C. granati on the stem were significantly larger than those caused by other species. In
addition, all plants inoculated with C. granati displayed wilting symptoms, congruent with field
observations. The necrotic lesions caused by N. parvum appeared depressed due to the collapse of
affected inner bark on xylem tissues.
No significant difference occurred among necrotic lesion sizes caused by P. palmivora, P.
pseudocryptogea and P. vexans at the collar, although the longest lesions were detected on seedlings
inoculated with P. palmivora (Fig. 2b). It is interesting to report that all the plants inoculated with P.
palmivora showed leaf chlorosis.
Control seedlings inoculated with sterile PDA or CA plugs remained symptomless (Fig. 1u, v). All
seven inoculated species were successfully re-isolated from the margin of symptomatic tissues of
inoculated plants, thus fulfilling Koch’s postulates.

Discussion

Results obtained have allowed us to clarify the aetiology of serious disease symptoms occurring on
pomegranate in Veneto (north-eastern Italy). In recent years, several studies have been carried out
throughout the world about postharvest fruit rots, shoot blights, branch cankers and root rots agents
on pomegranate (Pollastro et al., 2016; Markakis et al., 2017; Jabnoun-Khiareddine et al., 2018;
Lennox et al., 2018; Kurbetli et al., 2020). However, relatively few of these studies have reported
the occurrence of two or more symptoms or pathogens in the same plant or orchard. In this study a

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complex symptomatology and aetiology was observed in both orchards. Pomegranate plants were
characterized by different canopy and root disease symptoms.
						




Fig. 2 - Mean lesion length (cm) caused by Botryosphaeria dothidea (strain MEL6), Coniella granati (MEL2), Cytospora
punicae (MP4a) and Neofusicoccum parvum (MEL4) on the stem (A) and Phytophthora palmivora (MP24), Phytophtho-
ra pseudocryptogea (MP1) and Phytopythium vexans (MP43) at the collar (B) after 90 days on pomegranate seedlings.
Error bars represent the standard deviations from the mean. Values with the same letter above the bar do not differ signifi-
cantly at P = 0.05 according to LSD multiple range test.

Using comparisons of ITS sequence data combined with morphological and cultural features, eight
species belonging to seven genera and five families were identified from a collection of symptomatic
pomegranate samples. These included B. dothidea and N. parvum (Botryosphaeriaceae), D.
eres (Diaporthaceae), C. granati (Schizoparmaceae), C. punicae (Valsaceae), P. palmivora, P.
pseudocryptogea and P. vexans (Peronosporaceae). These findings show that the diversity of
pathogenic species associated with canopy and root symptoms of pomegranate is greater than
previously recognised. To our knowledge, B. dothidea, P. pseudocryptogea and P. vexans are reported
for the first time on pomegranate in Italy.
The pathogenicity of all main species obtained in this study was confirmed through an inoculation
experiment. All species tested, except C. punicae, were able to cause necrotic lesions and other canopy
symptoms. In particular, the isolate of C. granati, N. parvum and P. palmivora proved to be highly
aggressive. All these three species have recently been reported on pomegranate in different countries
worldwide. In particular, C. granati has been reported in California (Michailides et al., 2010), Spain
(Palou et al., 2010), Iran (Mirabolfathy et al., 2012), Turkey (Celiker et al., 2012), China (Chen et al.,
2014), Greece (Thomidis, 2015) and Italy (Pollastro et al., 2016) as an agent of postharvest fruit rots,
shoot blights, branch and stem cankers.
In this study C. granati was the main species associated with branch cankers and canopy dieback.
Interestingly, the plants positive to C. granati were also positive to P. palmivora, a plurivorous
pathogen recently reported on pomegranate in Greece and Turkey (Markakis et al., 2017; Kurbetli
et al., 2020). Phytophthora palmivora is one of the most important Phytophthora species in tropical
and sub-tropical regions. It is responsible for severe diseases on economically important tropical
crops such as cocoa, citrus, durian, jackfruit, oil palm, papaya and rubber tree (Erwin and Ribeiro,
1996; Guest, 2007; Borines et al., 2014; Peter and Chandramohan 2014). Annual losses due to disease
outbreaks in these crops impact for over US$ 2 billion (Derevnina et al., 2016; Torres et al., 2016). The
widespread presence of this pantropical pathogen in northern Italy highlights its broad adaptability to
different climatic conditions. Given the co-occurrence of these two aggressive pathogens in most of
the plants examined a synergistic interaction is highly plausible.
At the same time, several of the examined plants were affected by two or three species of canopy
and root pathogens suggesting a multitrophic interaction. In particular, in the site 1 N. parvun was
isolated together with B. dothidea and P. pseudocryptogea. The co-occurrence of Botryosphaeriaceae
and Phytophthora species on the same plants has also been detected in other pathosystems (Maloney

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et al., 2004; Linaldeddu et al., 2014, 2019). Recently, the presence of multiple Phytophthora species
associated with severe decline and mortality of pomegranate trees has been reported in Turkey
(Kurbetli et al., 2020).
Another interesting aspect of this research concerns C. punicae, which did not cause any disease
symptom on inoculated seedlings in the pathogenicity assay, although it was successfully re-isolated
from all plants. Cytospora punicae was detected from the same branch cankers affected by C. granati,
which in the pathogenicity test has reproduced the symptoms observed in nature. This suggests an
endophytic behaviour of C. punicae on pomegranate. This result is in contrast with the recent report
of C. punicae as a canker agent on pomegranate in the United States (Peduto et al., 2014) and Tunisia
(Triki et al., 2015).
This study represents the most comprehensive investigation of pomegranate-causing agents to date
in Italy. The complex etiological scenario which emerged in this study indicates that to develop
adequate management strategies to control emerging pomegranate diseases an accurate diagnosis is
necessary on all plant organs. Still, it is not clear the origin of this high diversity of pathogens present
in only two orchards.


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