1. Introduction

The green areas and urban natural elements are no strangers to the effects of global climate change, pollutants, biological invasion, and pests and diseases¹. Although inserted in an environment in which they are not the predominant element, sharing the space with abundant buildings and other services typical of cities, the city trees integrate an ecosystem with its own peculiarities². The urban forestry offers numerous benefits to the population, such as reduction of atmospheric pollution, regulation of air temperature, mitigation of city noise, regulation of water cycles, generation of recreational spaces, and the positive impact on human health in addition to the beautification of the streets³⁾⁽⁴. The interaction between the different components of the urban system -trees, pedestrians, urban infrastructure, utility services, vehicular traffic and streets- must keep a delicate balance to its better use and maintenance². Otherwise, successive interference between trees and other urban components can lead to risky situations due to the intensive management of these to adapt to the urban space⁵. In this sense Coelho-Duarte and others⁵ researched the attributes correlated to the likelihood of failure of trees in urban parks of Montevideo (Uruguay), where visual risk of failure and sanitary assessment were included in the studied methods.

In a review paper about pests and pathogens of Platanus spp. in urban forestry in Europe, Tubby & Pérez-Sierra reported symptoms attributed to Ceratocystis platani, and Splanchnonema platani as some of the most noteworthy pathogens affecting plane trees in Europe, as well as Fimotiporia punctata and Inonotus rickii, that also could cause canker and decay on trees⁶. However, no references were found for deformations manifested as bark fissures and hypertrophies.

Ceratocystis platani is a fungus that causes stain canker that was reported for the first time in the United States by Jackson and Sleeth, cited by Panconessi in 1999⁷ and by Tsopelas and others in 2017⁸, and was introduced in Europe during the Second World War in infected wooden crates. This fungus penetrates host tissue through wounds, causing canker and staining the surrounding bark with a bluish coloration. As it colonizes the xylem, it causes defoliation and branch die-back. The disease is present in many states of the United States and in most of European countries⁶.

Splanchnonema platani is a fungal pathogen that produces damages in the upper surface of main branches and that evolves towards progressive death of tissues with the appearance of cracks. It results in branch death and increases the likelihood of branch failure⁶. Until now, neither Ceratocystis nor Splanchnonema have been reported in our country, although they have been investigated in recent works⁹. To the best of our knowledge no studies on plane tree diseases have been done in South America.

Frequent symptoms present in Platanus x acerifolia in Montevideo are trunk hypertrophies, generally associated with bark fissures⁹, and appearing with different severity degrees (SEV). However, they can compromise the entire trunk. No references were found regarding these symptoms in Platanus spp.

The objectives of this work were: i) to quantify the symptoms of disease present on trunks, ii) to identify the fungi associated with them, and iii) to evaluate the relationship between observed symptoms and attributes correlated to the likelihood of failure of Platanus x acerifolia street trees in Montevideo, Uruguay.

2. Materials and methods

All the plane trees of 10 randomly selected city blocks among 100 city blocks at the B municipality of Montevideo, Uruguay, were selected for evaluation (Figure 1).

Figure 1: Map of Uruguay (A); Montevideo aerial view (B), and location of the area under study where the blocks analyzed are indicated (C) 

For each Platanus x acerifolia tree, diameter at breast height (DBH), tree height, presence of symptoms and likelihood of failure attributes were evaluated according to Pokorny¹⁰⁾ (Table 1). Additionally, information about the phytosanitary status according to observed symptoms¹¹, crown size, density, and presence of epicormic shoots (ES)¹² sidewalk lift (SL), and defects such as tree lean (TL), cracks, cavities, dead and hanging branches, pruning wounds, man-made damages, and severed, damaged or girdling roots (Table 1) were also registered. Presence of cankers, bark with reddish patches (reddish bark), or with black patches (black bark), sap oozing, hypertrophies (H) and bark fissures (BF) were registered. The incidences of each symptom separately, of the syndrome of bark fissures and hypertrophies, and of the cankers and reddish bark were calculated as the proportion of plants that showed them (number of trees with symptoms/total number of trees). The severity (SEV) was estimated from the affected proportion of trunk or branches and the position of the tree where symptoms were observed. For each symptom four levels of affected circumference (0%,<25%, 25-50% and >50%) and three different heights (tree divided in thirds: from the bottom to the first half of the trunk is the 1^st portion, the second half the 2^nd portion, and the crown the 3^rd portion) were combined to build four levels of SEV (healthy, slight, moderate, and serious) (Table 2). The Severity Main Index (SMI) was calculated according to the following formula:

were Nº₀ is the number of trees without symptoms; Nº₁ is the number of trees with slight SEV; Nº₂ is the number of trees with moderate SEV; Nº₃ is the number of trees with serious SEV; 0, 1, 2 and 3 are the SEV levels (Table 2).

The relationship between symptoms SEV and the severity categories (SCAT) of defects correlated to those likelihood of failure attributes was studied by estimating the Spearman correlation (p<0.05) to determine which of the former should be prioritized to treat and prevent.

Samples of vegetal tissue were taken from the transition zone between the healthy and the symptomatic parts of cankers. Samples were taken at three depths: superficially at the bark level, intermediate from phloem under the bark, and from heartwood (60-100 mm inside the trunk), by using a Pressler drill with a 4-mm-diameter bit, and also from a wooden roll of an already died tree. Each sample was carefully inspected to evaluate the presence of signs. Fragments of approximately 9 mm² of tissue were sterilized one minute in alcohol 96°, rinsed in distilled sterilized water and left to dry on sterilized paper in flux before being sown in potato dextrose agar growing medium (PDA, Oxoid Ltd., Hampshire, England). After a week of incubation at 24°C, the developed fungal colonies were examined: morphotypes and frequency of appearance (number of records of each morphotype in relation to the number of colonies developed) were registered. Some of the morphotypes were incubated under 12 hours of UV light A with 350 nm of wavelength to stimulate the development of reproductive structures that allow identifying the genus. For morphological identification at the genus level, fungal identification keys were used¹³⁾⁽¹⁴.

Table 1: Severity categories (SCAT) of factors associated with likelihood of failure 

Adapted from: (*)Vallejos¹¹; (**)Sepúlveda and Johnstone¹²; (***)Pokorny¹⁰

Table 2: Severity (SEV) levels to evaluate symptoms 

After identification, the isolates were included in the collection of the Plant Protection Department of the Agronomy School, University of the Republic.

For representative colonies of the more frequent morphotypes a DNA extraction from 10-days-old mycelia was performed following the protocol described by Paolocci and others¹⁵ with some modifications. They were macerated with lysis buffer (100ul/ml TRIS, HCl 200 mM pH7, 100ul/ml NaCl 250 mM, 100 ul/ml SDS 0,5%, 100ul/ml EDTA 25 mM + NaOH 10%, 600ul bidistilled water) and placed for 2 hours at -20°C. After that, mycelia were crushed with sterilized micropestle and treated with the lysis buffer, sodium chloride and isopropanol (-20°C) with the corresponding homogenizations, temperature incubation and centrifugations. Subsequently, the supernatant was discarded, and the pellet containing was rinsed with 70% alcohol, treated with a buffer TE (10 mM TRIS, HCl pH7.4 + 1mM EDTA pH8) and stored at -20°C.

The DNA was amplified by performing polymerasechain reaction (PCR) using a PTC-100 Peltier Thermal Cycler. Based on the morphological identification to genus level, different DNA regions were selected for amplifications. The internal transcribed spacer of the ribosomal DNA (ITS) was amplified for some isolates that could not be identified by morphology, since this region is universal for the Fungi kingdom¹⁶. For those isolates for which the genus could be identified by morphology, other primers recommended in the literature were used. For example, amplification of the region corresponding to glyceraldehyde-3-phosphate dehydrogenase genes (GAPDH) was performed for isolates within Colletotrichum genus¹⁷, and the elongation factor 1-α gene (EF-1α) was amplified for isolates within Botryosphaeria¹⁸⁾⁽¹⁹ and Pestalotiopsis genus²⁰. Details of the specific primers and amplification cycles can be found in Table 3. PCR products were analyzed on 1.5% agarose gels stained with GelRedTM and visualized in a transilluminator under UV light. Gene Ruler plus 100 bp DNA (Thermo, Lithuania) was used as a molecular marker. PCR products were purified and sequenced using only forward primers at Macrogen Inc., Seoul, Korea. The sequences were aligned with the ClustalW program with sequences of the gen bank. For species identifications the sequences were compared with the subjected to BLAST, those deposited in NCBI GenBank on the basis of percent identity values and query coverage.

Table 3: Primers and cycles used for fungal DNA amplification 

3. Results

From the 202 randomly selected trees, 197 were evaluated, because some specimens were located in construction areas where complete observations were not possible.

The incidence and SEV main index (SMI) of each symptom and of the association between cankers and reddish bark, and between bark fissure and hypertrophy are shown in Table 4; and it may be noticed that the highest values of incidence and SEV main index correspond to bark fissures and hypertrophy. The appearance of some mentioned symptoms is shown in Figure 2.

Figure 2: Recorded symptoms. Cankers (A, B); stretch marks and hypertrophies (C); stretch marks (D) reddish bark (E); black bark (F) 

The highest correlation was observed between bark fissures and hypertrophies; then these symptoms were also considered together (Table 5).

The overall phytosanitary status¹³ had the highest significant (p<0.05) correlation with SEV of bark fissures, hypertrophies and both symptoms considered together (Table 5). That variable also presented positive and significant correlation with cavities, man-made damage, dead branches, reddish bark, and the symptom of reddish bark and cankers considered together. The reddish bark only had significant correlation with dead branches, while the reddish bark and cankers considered together also had a significant relationship with crown density. The SEV of black bark showed a significant and positive correlation with the SCAT of pruning wounds, SCAT cavities, SCAT man-made damages and SEV bark fissures.

Table 4: Incidence and severity main index of the symptoms recorded 

Table 5: Significant correlations between recorded variables 

¹BF: bark fissures;² H: hypertrophies. Non-significant correlations are not shown.

The SEV of the bark fissures, hypertrophies and of both considered together had significant and positive correlations with the category of severity (SCAT) of pruning wounds and man-made damages. Neither significant correlation with the sidewalk lift was not found, nor in the case of the epicormic shoots with none of the recorded symptoms.

From the tissue samples obtained from cankers and reddish bark, 59 fungal colonies and 10 morphotypes were obtained. Among them, the 3 most frequent were 42 colonies out of the 59. Based on morphological characters (mycelium colour and texture, type of spores, presence/absence of fruiting bodies, etc.) genera such as Pestalotiopsis, Diplodia, Epicoccum and Phomopsis were identified (Figure 3).

Figure 3: Reproductive structures observed. Diplodia sp. sporodochium (A); conidia of Pestalotiopsis sp. (B); pycnidium and tendrils of Phomopsis sp. (C) 

Samples from heartwood showed rots with pink and grey coloration (Figure 4). From these samples, 12 of the obtained colonies were identified as Colletotrichum acutatum, and the remaining 3 to the genera Ophiostoma, Nigrospora and Aureobasidium.

From the analysis of sequences several genera were confirmed, and the following species were identified: Neofusicoccum parvum, Diplodia mutila, Diplodia pseudoseriata, Pestalotiopsis biciliata, Pestalotiopsis rhodomyrtus and Colletotrichum acutatum (Table 6). The most frequent morphotypes corresponded to Pestalotiopsis spp., Diplodia spp. and Neofusicoccum spp. The genera Phomopsis, Phoma, Nigrospora, Aureobasidium, Epicoccum and Torula were identified in a very low frequency.

Figure 4: Wood samples with rot with pinkish and greyish coloration 

Table 6: Identified species with sequence analysis 

Table 7: Frequency of identified fungi from bark and wood 

4. Discussion

Although some of the symptoms evaluated in this study resemble those previously described as being caused by Ceratocystis platani or by Splanchnonema platani^{6)(7)(8)(9)(21)(22)(23}, none of these pathogens were previously reported in our region neither isolated from the samples processed in this work. Similarly, Pelleret and others²⁴, studying plane trees with symptoms like those caused by C. platani (cankers and dieback, trunk, and branch necrosis), mainly isolated Neofusicoccum parvum and Diplodia pseudoseriata among others Botryosphaeriaceae, and isolated Ceratocystis platani only from 2 trees (out of 6 symptomatic trees).

Considering the results of Turco and others²⁵ and Kurbetli and others²⁶, it can be observed that the mentioned symptoms can also be caused by other genera, those ones belonging to the Botryosphaeriaceae family. These include many genera that are also related to branch and trunk canker, necrosis, wood discoloration, branch dieback on fruit trees^19)(27)(28 and forest trees^{29)(30)(31)(32)(33)(34}. Species belonging to this family are also reported as endophytes³⁵⁾⁽³⁶. Pelleret and others²⁴ hypothesize that these Botryosphaeriaceae species could be responsible for the observed cankers on plane trees, while other fungal species could contribute to the dieback symptoms. Similarly to Pelleret and others²⁴, during this study Diplodia mutila and Neofusicoccum parvum were isolated from symptomatic tissue of Platanus x acerifolia. D. mutila was reported causing die-back, cankers, necrosis, dead branches and twigs in Quercus³², dieback in grapevine²⁸, cankers and dieback in araucarias³⁰, and cankers, dieback, and internal necrosis in walnut³³. Diplodia pseudoseriata also was reported causing cankers, branch dieback and fruit rots in apples¹⁸, and cankers and branch dieback in citrus³⁷.

Neofusicoccum parvum was reported as associated to cankers and dieback in eucalyptus in Spain and in Mexico³²⁾⁽³³, to cankers and die back in grapevine and sequoia²⁸⁾⁽³⁴, and was also reported together with D. pseudoseriata causing cankers and branch dieback in citrus²⁷.

Regarding the isolation of Pestalotiopsis spp. and of Colletotrichum spp., no previous report has been found about their pathogenicity on Platanus spp.³⁸; however, Leite and others³⁹ found Pestalotia spp., the closest genus related to Pestalotiopsis and that belongs to the Pestalotia-Pestalotiopsis complex⁴⁰, as epiphytes in Platanus orientalis bark in very low frequency, but working with different culture media than the one used in the present work.

Among Colletotrichum, there are species considered nonpathogenic endophytes, and other that are considered to host specific necrotrophic pathogens. Several fungal lineages change from endophytes to pathogens on short time scales, including Colletotrichum species⁴¹⁾⁽⁴². Although the species C. gloeosporioides is the most mentioned as pathogenic associated to senescent tissues, at present, probably due to advances in identification techniques, it is C. acutatum the one that appears to predominate as pathogenic, and in some cases, as in citrus and coffee, associated to symptoms in flowers causing fruit drop⁴². More recently, C. gloeosporioides and C. karstii associated to dieback of citrus branches have been reported⁴³.

Regarding the genus Pestalotiopsis, belonging to the Pestalotiopsidaceae class, it includes species recognized as pathogens⁴⁴⁾⁽⁴⁵, but also endophytes, and, lately, species that produce metabolites for medicinal use are being studied⁴⁶⁾⁽⁴⁷.

The possibility that several of the species identified in this work were endophytes³⁵⁾⁽³⁶ raises questions regarding their role: so, it is necessary to accomplish Koch postulates, and to analyse healthy bark and wood tissue to determine if they performed as endophytes or as pathogens.

The bark fissures and hypertrophy are associated to each other and to the black bark (Table 5). At the same time, they have positive correlation with the pruning wounds and with the man-made damages, suggesting that it is an urban disorder. Although the cause of bark fissures and hypertrophy considered together is not known yet, the tree workers and managers from Montevideo do not associate it with failure of branches or trees. This coincides with the fact that no significant correlation has been found between these symptoms and the presence of dead or hanging branches, cracks and cavities, indicators associated to the likelihood of failure of the tree or parts of it⁵⁾⁽¹². No pathogen sign was observed, but the symptoms are very similar to those caused by Xanthomonas populi pv. Populi⁴⁸ in Populus, so more studies must be undertaken to determine if the ones recorded in the trees studied in this work could have been generated by any Xanthomonas spp.

Although the phytosanitary status was correlated with bark fissures and hypertrophy, in the present work insufficient evidence was found to prove that these deformations affect the health of the specimens and could not be a further damage than aesthetic.

The significant correlation of the cankers and reddish bark with the reduction of crown density, as well as of the reddish bark with the greater presence of dead branches could indicate that these symptoms may be the cause of dieback, an indicator associated to the likelihood of failure of branches⁵. However, considering the correlation values (Table 5), it is necessary to deepen the study of this relationship.

5. Conclusions

The fungal genera more frequently isolated were Pestalotiopsis, Diplodia, Neofusicoccum and Colletotrichum.

The cankers and reddish bark affect the urban trees shadow; however they are not associated to each other.

The symptoms attributed in scientific literature to Ceratocystis platani also can be caused by other fungal pathogens.

The black bark symptom, and bark fissures and hypertrophy considered together appeared associated to the presence of pruning wounds and man-made damages, so the latter may be the cause of the formers or act predisposing the trees to express them.