Neopestalotiopsis siciliana sp. nov. and N. rosae Causing Stem Lesion and Dieback on Avocado Plants in Italy

Avocado (Persea americana) represents an important emerging tropical crop in Italy, especially in the southern regions. In this study, young plants of avocado showing symptoms of stem and wood lesion, and dieback, were investigated. Isolations from symptomatic tissues consistently yielded colonies of Neopestalotiopsis-like species. The characterization of representative isolates was based on the observation of morphological characters, the effect of temperature on mycelial growth rate, and on the sequencing of three different gene regions, specifically ITS, TEF1, and TUB2. Phylogenetic analyses were conducted based on maximum parsimony and maximum likelihood approaches. The results showed the presence of two species, viz. Neopestalotiopsis rosae and N. siciliana, the latter of which is here described as a new species. Pathogenicity tests were conducted using the mycelial plug technique on young potted avocado trees for both Neopestalotiopsis species. The results showed that both species were pathogenic to avocado. This study represents the first report of these two species affecting avocado and results in the description of a new species within the genus Neopestalotiopsis. Based on phylogeny, Pestalotiopsis coffeae-arabicae is combined in Neopestalotiopsis.


Introduction
Avocado (Persea americana Mill.) is a tree native to Central America and is widely cultivated, especially in tropical and subtropical regions. In the recent years, the consumption and new plantings of this tropical fruit are increasing world-wide. Global top producers (1000 metric tons unit, year 2020) include Mexico (2390), Colombia (876), and the Dominican Republic (676) [1]. Of European countries, Spain is the main avocado producer (99), followed by Greece (10) [1]. In southern Italy, specifically in Sicily, avocado represents an emerging crop in terms of economic opportunity for the growers. In recent years, an increasing consumer interest towards tropical fruits has been observed. Within this new trend, avocado fruit presents great potential due to its high nutritional value and peculiar quality characteristics to achieve requirements desired by consumers [2]. In Italy, since this crop is emerging, few studies have been conducted on the phytopathological situation. Regarding the fungal diseases affecting avocado, several fungal taxa have been reported associated with different symptoms [3], and some of those pose a severe threat for its production around the world. Among these, Phytophthora cinnamomi is considered the most important and widely spread pathogen of avocado, causing significant economic losses [4]. Rosellinia necatrix, the causal agent of white root rot, is a serious threat in the Mediterranean area and is considered the main cause of avocado losses in Spain [5], and recently it has also been reported in Italy [6]. In Italy, as well as around the world [7], several species belonging to the Nectriaceae family (i.e., Cylindrocladiella peruviana, Ilyonectria macrodidyma, and Pleiocarpon algeriense) have been studied in the last few years, and have been associated with root and crown rot [8,9]. Several fungal species belonging to Botryosphaeriaceae and Diaporthaceae families are known to be causal agents of branch canker and fruit stem-end rot on avocado [10][11][12][13]. In addition, in 2018, a new species named Neocosmospora perseae was described as causing trunk cankers in Italy and more recently in Greece [14,15]. Colletotrichum spp. are reported as important pre-and postharvest pathogens [16][17][18][19]. Moreover, pestalotioid fungi, known as major agents of leaf spot diseases, were also reported on avocado [20][21][22]. During December of 2020, surveys conducted in a commercial avocado orchard in Sicily (Italy) revealed the presence of young plants showing external symptoms of dieback and stem lesions on scion at the grafting point or slightly above. Since avocado is considered an emerging crop in Italy, especially in the southern regions, it is crucial to investigate the etiology of the diseases that could represent an important limiting factor. The aim of the present study was to investigate the etiology of the symptoms observed in the field and to identify the causal fungal agents to species by morphology and molecular data.

Isolation and Morphological Characterization
Samples were collected in the field on approximately 20 plants of Persea americana cv. "Hass" grafted on "Zutano", randomly selected, and were brought to the Plant Pathology laboratory of the Department of Agriculture, Food, and Environment at the University of Catania for further investigations. One hundred small sections (5 mm × 5 mm) of the stems were surface disinfected for 1 min in 1.5% sodium hypochlorite (NaOCl), rinsed in sterile distilled water, dried on sterile absorbent paper, and placed on potato dextrose agar (PDA; Lickson, Vicari, Italy) amended with 100 mg/liter of streptomycin sulphate (Sigma-Aldrich, St. Louis, MO, USA) to prevent bacterial growth, and then incubated at 25 ± 1 • C for five to seven days. Single-spore isolates were obtained from conidia produced in pure cultures grown on PDA. To determine the effect of temperature on mycelial growth and the optimal growth temperature, the representative isolates AC46 and AC50 were cultured on PDA for further assays. After seven days of incubation at 25 • C, 5 mm diam. mycelial plugs were transferred from the edge of the colonies to the center of PDA Petri plates. The plates were incubated at 5-10-15-20-25-30-35 • C in the dark. Three Petri plates were used for each temperature as replicates. The experiment was repeated once. After seven days of incubation, two perpendicular diameters of the colonies were measured with a scale ruler. The isolates used in this study are maintained in the culture collection of the Department of Agriculture, Food, and Environment, University of Catania. Moreover, representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, the Netherlands, and dried sporulating cultures were deposited as vouchers in the fungarium of the Department of Botany and Biodiversity Research, University of Vienna (WU-MYC).
Study of macromorphology of conidiomata was performed by using a Nikon SMZ 1500 stereomicroscope equipped with a Nikon DS-U2 digital camera or with a Keyence VHX-6000 digital microscope (Mechelen, Belgium). Microscopic preparations were mounted in water. For light microscopy, a Zeiss Axio Imager.A1 compound microscope (Oberkochen, Germany), equipped with Nomarski differential interference contrast (DIC) optics and a Zeiss Axiocam 506 color digital camera, was used. Photographs and measurements were taken by using the NIS-Elements D v. 3.0 or Zeiss ZEN Blue Edition software. For certain images of conidia, the stacking software Zerene Stacker version 1.04 (Zerene Systems LLC, Richland, WA, USA) was used. Measurements are reported as maxima and minima in parentheses and the mean plus and minus the standard deviation of a number of measurements given in parentheses.

DNA Extraction, PCR, and Phylogenetic Analysis
The genomic DNA was extracted from surface mycelium scraped off from pure cultures, using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA). The following three loci were amplified and sequenced: the complete internal  Sequence alignments for phylogenetic analyses were produced with the server version of MAFFT (http://mafft.cbrc.jp/alignment/server/, accessed on 22 March 2022), and checked and refined using BioEdit v. 7.2.6 [57]. The ITS rDNA, TEF1, and TUB2 matrices were combined for subsequent phylogenetic analyses, and the combined data matrix contained 2265 characters (545 nucleotides of ITS, 900 nucleotides of TEF1, and 820 nucleotides of TUB2). Maximum likelihood (ML) analyses were performed with RAxML [58], as implemented in raxmlGUI 1.3 [59], using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates. The matrix was partitioned for the different gene regions. For evaluation and discussion of bootstrap support, values below 70% were considered low, between 70 and 90% medium/moderate, and above 90% high and 100% maximum. Maximum parsimony (MP) bootstrap analyses were performed with PAUP v. 4.0a169 [60], with 1000 bootstrap replicates using five rounds of heuristic search replicates with random addition of sequences and subsequent TBR branch swapping (MULTREES option in effect, steepest descent option not in effect, COLLAPSE command set to MINBRLEN, and each replicate limited to 1 million rearrangements) during each bootstrap replicate. All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data; and the COLLAPSE command was set to minbrlen.

Pathogenicity Test
Pathogenicity tests were carried out by artificial inoculations using the isolates AC50 (N. rosae) and AC46 (N. siciliana). Three potted 1-year-old plants of avocado cv. "Hass" grafted on "Zutano" were inoculated with each isolate. Inoculations were made on the stem after removing a piece of bark with a sterile scalpel blade, placing a mycelial plug (0.3 cm 2 ) from a 15-day-old culture of each isolate onto the wound and covering it with Parafilm ® (American National Can, Chicago, IL, USA) to prevent desiccation. The same number of plants was inoculated with sterile PDA plugs to serve as control. All the inoculated plants were grown in a growth chamber with a 12 h photoperiod and maintained at 25 ± 1 • C. The inoculated plants were monitored weekly for development of symptoms, and a final assessment was conducted 50 days after the inoculations. Re-isolations were performed as described above to fulfill Koch's postulates.

Data Analysis
Data derived from the effect of temperature on mycelial growth rate assay and the lesion length measurements were analyzed in Statistix 10 (Analytical Software 2013) [61]. For analysis of the effect of temperature on mycelial growth, variances of the two assays were tested for the homogeneity using Levene's test and then combined in one dataset. Data of the mycelial growth were first transformed to radial growth rate (cm day −1 ) and then a nonlinear regression adjustment of the dataset was applied through the generalized Analytis β model, using the equation described by Moral et al. [62]. Optimum growth temperature was also calculated according to the equation provided by the same authors [62]. For pathogenicity test, analysis of variance (ANOVA) of the lesions length was performed and the mean differences were compared with Fisher's protected least significance difference (LSD) test at α = 0.05.

Isolation and Morphological Characterization
The disease was observed in a commercial avocado orchard located in Giarre (Catania province) on young plants (two years old, 2-3 months after transplanting). Symptoms observed in the field included stem lesions, wood discoloration with brownish streaking, bark cracking, and dieback. Necrotic lesions were characterized by a shrinkage of the affected tissues and internally the wood appeared darkened and dry (Figure 1). Internal lesions started more frequently from the grafting point. The rootstock showed no symptoms. Isolations frequently yielded Neopestalotiopsis-like fungi. A total of eight single-spore isolates were collected and kept in our fungal collection. The highest growth rate for the isolate AC46 (1.13 cm day −1 ) was observed at 25 • C. According to the Analytis β model, the optimal growth temperature resulted at 24.6 • C. After 7 days of incubation, no mycelial growth was observed at 35 • C. Isolate AC50 showed the highest growth rate (1.06 cm day −1 ) at 25 • C, and the optimal growth temperature resulted at 21.9 • C. All results of the effects of temperature on mycelial growth rate are shown in Figures 2 and 3. toms observed in the field included stem lesions, wood discoloration with brownish streaking, bark cracking, and dieback. Necrotic lesions were characterized by a shrinkage of the affected tissues and internally the wood appeared darkened and dry (Figure 1). Internal lesions started more frequently from the grafting point. The rootstock showed no symptoms. Isolations frequently yielded Neopestalotiopsis-like fungi. A total of eight single-spore isolates were collected and kept in our fungal collection. The highest growth rate for the isolate AC46 (1.13 cm day −1 ) was observed at 25 °C. According to the Analytis β model, the optimal growth temperature resulted at 24.6 °C. After 7 days of incubation, no mycelial growth was observed at 35 °C. Isolate AC50 showed the highest growth rate (1.06 cm day −1 ) at 25 °C, and the optimal growth temperature resulted at 21.9 °C. All results of the effects of temperature on mycelial growth rate are shown in Figures 2 and 3.

Phylogenetic Analysis
PCR amplification of the ITS, TEF1, and TUB2 generated 549, 549-550, and 957 bp fragments, respectively. Of the 2261 characters included in the phylogenetic analyses, 334 were parsimony informative (58 from the ITS, 153 from TEF1, and 123 from TUB2). The best ML tree (−lnL = 8699.596) revealed by RAxML is shown as a phylogram in Figure 4. While backbone support of deeper nodes was mostly absent, several terminal nodes received medium to high support. Of the four Neopestalotiopsis isolates of the current study, one was placed within the N. rosae clade, while the other three isolates were contained within a moderately supported clade together with four unnamed Japanese isolates from Eriobotrya japonica. The latter clade was further subdivided into two subclades: a highly supported subclade containing the three isolates of the present study and one isolate from Eriobotrya japonica, and a second, moderately supported subclade containing the residual isolates from Eriobotrya japonica.

Phylogenetic Analysis
PCR amplification of the ITS, TEF1, and TUB2 generated 549, 549-550, and 957 bp fragments, respectively. Of the 2261 characters included in the phylogenetic analyses, 334 were parsimony informative (58 from the ITS, 153 from TEF1, and 123 from TUB2). The best ML tree (−lnL = 8699.596) revealed by RAxML is shown as a phylogram in Figure 4. While backbone support of deeper nodes was mostly absent, several terminal nodes received medium to high support. Of the four Neopestalotiopsis isolates of the current study, one was placed within the N. rosae clade, while the other three isolates were contained within a moderately supported clade together with four unnamed Japanese isolates from Eriobotrya japonica. The latter clade was further subdivided into two subclades: a highly supported subclade containing the three isolates of the present study and one isolate from Eriobotrya japonica, and a second, moderately supported subclade containing the residual isolates from Eriobotrya japonica. Strains marked by an asterisk (*) represent ex-type strains. ML and MP bootstrap support above 50% are given above or below the branches. The broken branches were scaled to one tenth.
Habitat Notes. The phylogenetic analyses revealed a highly supported, distinct phylogenetic position within Neopestalotiopsis, confirming its status as a new species. Remarkably, it clusters with four unnamed Neopestalotiopsis accessions isolated from the leaves and fruits of Eriobotrya japonica in Japan [55]. While one of these strains has sequences identical to our isolates, with which it clustered within a highly supported subclade, the three other strains from Eriobotrya form a sister clade to the former ( Figure 4). As there are some molecular differences between these two subclades, it is yet unclear whether one or two species are involved; considering the uncertainties in species circumscription in the genus and the lack of morphological data, we here maintain these isolates as Neopestalotiopsis sp. As usually observed in Neopestalotiopsis, it is impossible to identify N. siciliana by conidial morphology alone, and sequence data are necessary for reliable species identification.

Discussion
The results of this study confirm the presence of Neopestalotiopsis species causing disease on young avocado plants in southern Italy. The fungal species obtained from symptomatic tissues were identified based on the morphological characteristics and molecular phylogenetic analyses of the ITS, TEF1, and TUB2 gene regions. The phylogenetic analyses showed that two species are involved in avocado stem and wood lesions, resulting in the dieback of the plants. Of the four isolates sequenced, one was identified as N. rosae, while another three isolates formed a clade distinct from the other described Neopestalotiopsis species, which is therefore here described as a new species, N. siciliana. Remarkably, these three isolates were phylogenetically close to four unnamed Neopestalotiopsis strains isolated from Eriobotrya japonica in Japan [55]; one even had identical sequences to our isolates. This demonstrates that N. siciliana is widespread and has a wider host range. There are a few reports from avocados attributable to the genus Neopestalotiopsis, for which ITS sequences are available. Valencia et al. [20] recorded N. clavispora (as Pestalotiopsis clavispora; ITS sequence HQ659767) as a causal agent of post-harvest stem-end rot in Chile, while Shetty et al. [69] identified one of their endophytic isolates from organically grown avocado trees in South Florida as Neopestalotiopsis foedans (ITS sequence KU593530). A sequence comparison of the ITS sequences with our matrix showed that the ITS sequence HQ659767 was identical and that KU593530 was almost identical (one gap difference) to our isolate AC50 representing N. rosae. While this could indicate that N. rosae is regularly found on avocado, it needs to be noted that the ITS alone is not suitable for species identification, as several species (e.g., N. hispanica, N. longiappendiculata, N. mesopotamica, N. scalabiensis, and N. vaccinii) have ITS sequences identical to those of N. rosae. Therefore, the species identity of these avocado isolates remains uncertain in the lack of TEF1 and TUB2 sequences.
Pestalotiopsis sensu lato was recently revised by Maharachchikumbura et al. [21] and segregated into three distinct genera, viz. Pestalotiopsis, Pseudopestalotiopsis, and Neopestalotiopsis. During our field surveys, we often encountered young plants of avocado showing stunted growth. Monitoring the plants after the transplant from the nurseries to the open field, it was noticed that some of these were not able to survive. Deeper observations of the internal tissues revealed frequent necrosis and cankers at the grafting point. Likely, propagation processes represent relevant infection courts for pestalotioid fungi. Most of the avocado plants transplanted in Sicily derive from Spanish nurseries where the propagation steps are performed. It is not unusual that symptoms such as stunting, shoot blight, and cankers observed by the growers after the first years from the transplant in the open field are the results of previous infections that had occurred in the nurseries, especially during the grafting. In fact, wounds and injuries are crucial for penetration of the host tissue and subsequent development of the infection, especially for pestalotioid species [70]. Our observations are indeed in accordance with other reports. In China, 30% of symptomatic avocado plants in a nursery plantation showed the presence of Pestalotiopsis longiseta [71] and other authors also reported the presence of Pestalotiopsis spp. at the graft union in different hosts [72][73][74]. Species of these genera are widely distributed in tropical and temperature areas. This group of fungi is commonly found as endophytes and plant pathogens on different hosts, causing stem-end rot, stem and leaf blight, trunk disease, and cankers [21,75]. Previous investigation conducted in Sicily already reported the presence of pestalotioid fungi, including Pestalotiopsis clavispora and P. uvicola, causing diseases on tropical, as well as on ornamental, hosts [22,75]. This study represents a new step forward in the insight of this complex and still understudied group of fungi, especially within the genus of Neopestalotiopsis, providing new information on the ecology of these two species.

Conclusions
Two fungal species, Neopestalotiopsis rosae and N. siciliana sp. nov. are described and illustrated. These fungi were isolated from the stem tissues of diseased young avocado plants in Sicily, Italy. Pathogenicity tests were performed, and Koch's postulates were fulfilled. The result of this study provided new information regarding this still understudied group of phytopathogenic fungi and its wide host range. Neopestalotiopsis rosae and N. siciliana could be a new threat to the Italian avocado industry, especially in the southern regions where avocado represents an emerging crop. The presence of these species in the internal tissues at the graft union corroborates the fact that the propagation processes represent crucial steps to obtain healthy material. Further investigations are needed in order to ascertain the diffusion and epidemiology of these species in the Sicilian avocado orchards, and to evaluate the effective risk for the industry. Therefore, it will be important to carry out additional studies on the pathogenicity and susceptibility of the different cultivars of avocado in the future. To our knowledge, this is the first report of N. rosea and of the fungus here described as N. siciliana affecting avocado.

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.