Diversity, Abundance, and Distribution of Wood-Decay Fungi in Major Parks of Hong Kong

Wood-decay fungi are one of the major threats to the old and valuable trees in Hong Kong and constitute a main conservation and management challenge because they inhabit dead wood as well as living trees. The diversity, abundance, and distribution of wood-decay fungi associated with standing trees and stumps in four different parks of Hong Kong, including Hong Kong Park, Hong Kong Zoological and Botanical Garden, Kowloon Park, and Hong Kong Observatory Grounds, were investigated. Around 4430 trees were examined, and 52 fungal samples were obtained from 44 trees. Twenty-eight species were identified from the samples and grouped into twelve families and eight orders. Phellinus noxius, Ganoderma gibbosum, and Auricularia polytricha were the most abundant species and occurred in three of the four parks. Most of the species were detected on old trees, indicating that older trees were more susceptible to wood-decay fungi than younger ones. More wood-decay fungal species were observed on Ficus microcarpa trees than on other tree species. These findings expanded the knowledge of wood-decay fungi in urban environments in Hong Kong and provided useful information for the conservation of old trees and the protection of human life and property from the danger of falling trees.


Introduction
Urban forests play a very important role in the urban ecological system. They provide shelter to urban animals, adsorb and accumulate anthropogenic pollutants, and can decontaminate the polluted soils and water through phytoremediation [1][2][3]. Well planned and managed urban forests can support high levels of biodiversity [4,5]. It is also important that urban forests add additional value to dwellings and people are willing to pay to protect urban forests [6,7]. This is especially true in cosmopolitan Hong Kong. Old trees are registered as Old and Valuable Trees (OVTs) for protection and management purposes. The old trees provide aesthetic value in addition to their ecological functions. However, urban forests are vulnerable to disturbance from weather and infection, and falling trees can be significant human hazards [8]. Failure of trees in urban forest is usually caused by microbial infection and lack of adequate soil depth because of poor soil conditions, excessive human activities, and lack of proper management. Wood-decay fungi are the most relevant and recognized pathogens to growing and dead trees.
Wood-decay fungi are capable of breaking down complex, high-molecular weight constituents in the plant cell wall into small molecules for assimilation by the fungi involved as well as other (http://www.hko.gov.hk/tide/marine/hko.htm, https://maps.google.com.hk/).

Field Sampling
The infected trees could be visually identified based on symptoms, such as dieback, defoliation, discoloration, hollows, or cavities on tree trunks. These trees were selected for disease diagnosis. Fungal fruiting bodies, if present, were sampled for further identification. Otherwise, the roots of suspected trees were dug out from the soil to check for fungal colonization, and wood tissues were sampled aseptically and sent back to the laboratory for further analysis.
Besides the infected trees, all the OVTs in these parks (except HKO) were all checked regardless of the status they displayed. Two rounds of samplings were carried out in May 2012 (for the wet

Field Sampling
The infected trees could be visually identified based on symptoms, such as dieback, defoliation, discoloration, hollows, or cavities on tree trunks. These trees were selected for disease diagnosis. Fungal fruiting bodies, if present, were sampled for further identification. Otherwise, the roots of suspected trees were dug out from the soil to check for fungal colonization, and wood tissues were sampled aseptically and sent back to the laboratory for further analysis.
Besides the infected trees, all the OVTs in these parks (except HKO) were all checked regardless of the status they displayed. Two rounds of samplings were carried out in May 2012 (for the wet season) and in November 2012 (for the dry season) to obtain as many samples as possible, especially of the non-perennial fruiting bodies.

Culturing and Isolation
Samples were transported back to the laboratory for processing within hours. The wood tissues with fungal mycelia were cut aseptically into several pieces (approximately 3 mm 3 ) under aseptic conditions. Each piece was submerged into 75% ethanol for 2 min, rinsed three times with sterile water, and placed on potato dextrose agar (PDA) (BD DifcoTM) plates adjusted with 0.5% streptomycin, then incubated at room temperature. Fungal hyphal tips were transferred to fresh PDA plates for isolation and purification.

PCR Amplification
Total genomic DNA of approximately 5 mg of fruiting bodies or pure isolates were extracted with an E.Z.N.A. ® Forensic DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the protocol provided for PCR amplification. One pair of universal primers ITS1F (5 -CTTGGTCATTTAGAGGA AGTAA-3 ) [24] and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) [25] was used to amplify the internal transcribed spacer (ITS) regions (including ITS1, 5.8S and ITS2) of the pure cultures or fruiting bodies. The PCR amplification reaction system included 5 µL of 5× PCR buffer, 2 µL of 25 mM MgCl 2 , 1 µL of 10 mM deoxyribonucleotide triphosphate (dNTPs), 1 µL of 10 µM of each primer (ITS1F and ITS4), approximately 10 ng template DNA, 0.15 µL of 5 U/µL GoTaq ® DNA Polymerase (Promega, Madison, WI, USA), 1 µL of 1% BSA (bovine serum albumin) (w/v) and finally made up to 25 µL with sterile water. The thermal cycling protocol included an initial denaturation at 95 • C for 3 min, followed by 32 cycles consisting of denaturation at 95 • C for 45 s, annealing at 52 • C for 30 s, extension at 72 • C for 45 s, and a final extension at 72 • C for 10 min. Amplified products were visualized on 1% agarose electrophoresis gels stained with GelRed TM Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA) to check for product purity and size. PCR products were purified with GFX TM PCR RNA and a Gel Band Purification Kit (Amersham Biosciences, Waltham, MA, USA). Purified PCR products were sequenced at the Genome Research Centre of The University of Hong Kong. The nucleotide sequences have been deposited in GenBank (accession numbers KU194304-KU194347).

Phylogenetic and Statistical Analysis
The obtained sequences were checked and revised with BioEdit v7.0.5.3 [26]. Basic Local Alignment Search Tool (BLAST) searches were performed using the GenBank database (http://www.ncbi.nlm.nih. gov). The sequences with highest similarity and coverage (Table S1) were downloaded for alignment using ClustalW in BioEdit and then conducted phylogenetic analysis [27]. The neighbor-joining tree with 1000 replications of bootstraps was built with the alignment in MEGA 5.05 after a minor manual adjustment [28]. To confirm the accuracy of the phylogenetic analysis, Bayesian Posterior Probability was calculated with BEAST v1.5.4 [29]. The best model for Bayesian analysis was selected with MrModeltest 2.2 [30]. Both branch support numbers were shown at the nodes of the neighbor-joining trees.
The isolation frequency of major wood-decay fungi was estimated by calculating the percentage of each fungus in all the samples collected in the parks (number of isolates/n, where n = 52). The occurrence rate of each species was calculated as the frequency of occurrence of a species in the four parks (e.g., P. noxius was detected in three out of four parks, and therefore the occurrence rate was 75%). To study the species assemblage of the wood-decay fungi in each park, relative occurrence of each wood-decay fungal species was calculated with the occurring frequency of each species in each park. The colonization rate of wood-decay fungi on old trees was compared with that on the younger trees. The colonization rate on OVTs and non-OVTs was compared with a paired t test in GraphPad Prism 5.0.

Morphological Characters
The identification of fungal fruiting bodies and fungal cultures was conducted mainly based on molecular techniques stated above, but morphological characters were also used to confirm the results from molecular methods. For morphological characteristics, pictorial guides were used, and microscopic observations were conducted following the authorized descriptions [12,20,[31][32][33]. Fruiting bodies were selectively dried in the drying oven (70 • C) and kept as specimens and cultural isolates were inoculated onto slants containing PDA medium and kept at 4 • C for storage.

Diversity of Wood-Decay Fungi in Parks
During this survey, around 4430 trees from four major parks in Hong Kong were examined. A total of 52 fungal isolates were obtained from 44 trees. The colonization rate of trees by wood-decay fungi was approximately 0.7% in the surveyed parks. The ITS rDNA PCR amplification, together with morphological characterization of the fruiting bodies and culture isolates, retrieved 28 fungal species, which were affiliated with 12 families from 8 orders ( Table 2).  The phylogeny based on multiple sequence alignment showed that the dominant wood-decay fungi detected in this study were affiliated with the orders of Hymenochaetales ( Figure 2) and Polyporales ( Figure 3). Within the order of Hymenochaetales, two genera, Phellinus and Fuscoporia, were identified and both are important pathogenic wood-decay fungi affiliated with the family of Hymenochaetaceae ( Figure 2). Within in the order of Polyporales, multiple fungal species were identified, and they belong to four families: Ganodermataceae, Polyporaceae, Phanerochaetaceae and Meripilaceae ( Figure 3).

Abundance and Distribution of Wood-Decay Fungi in Different Parks
Nine fungal species, accounting for 60% of the wood-decay fungi detected in this study were considered as most abundant wood-decay fungi in the four parks investigated (Figure 4). Among the nine fungal species, P. noxius was the most abundant, followed by G. gibbosum and A. polytricha. These three species were also detected in most parks.

Abundance and Distribution of Wood-Decay Fungi in Different Parks
Nine fungal species, accounting for 60% of the wood-decay fungi detected in this study were considered as most abundant wood-decay fungi in the four parks investigated (Figure 4). Among the nine fungal species, P. noxius was the most abundant, followed by G. gibbosum and A. polytricha. These three species were also detected in most parks.   To calculate the occurrence rate of each wood-decay fungal species in each park and on each tree species ( Figure 5 and Table 3), fungi that were reported as root-rot pathogens or could cause serious wood-decay were listed with species names while less significant fungal species or fungi with unknown causes were categorized as "others". According to the relative occurrence, KP hosted the largest number of wood-decay fungal species, followed by ZBG, and then HKO and HKP. In KP, in  To calculate the occurrence rate of each wood-decay fungal species in each park and on each tree species ( Figure 5 and Table 3), fungi that were reported as root-rot pathogens or could cause serious wood-decay were listed with species names while less significant fungal species or fungi with unknown causes were categorized as "others". According to the relative occurrence, KP hosted the largest number of wood-decay fungal species, followed by ZBG, and then HKO and HKP. In KP, in addition to eight significant wood-decay fungal species (about 50% of the total samples collected from KP), around 50% of the fungi detected were not known to be pathogens, suggesting that KP may have a higher diversity of wood-decay fungi. Most of the fungi were observed in more than one park, but some fungi were only detected in one park. For example, R. vinctus, G. webrianum, and F. senex were only detected in ZBG. In this study, the samples were collected from 13 living tree species and dead wood or wood stumps, fallen branches, and shrubs in the four parks. More wood-decay fungal species were detected on dead trees or wood stumps and F. microcarpa than the rest of the wood types (Table 3). To calculate the occurrence rate of each wood-decay fungal species in each park and on each tree species ( Figure 5 and Table 3), fungi that were reported as root-rot pathogens or could cause serious wood-decay were listed with species names while less significant fungal species or fungi with unknown causes were categorized as "others". According to the relative occurrence, KP hosted the largest number of wood-decay fungal species, followed by ZBG, and then HKO and HKP. In KP, in addition to eight significant wood-decay fungal species (about 50% of the total samples collected from KP), around 50% of the fungi detected were not known to be pathogens, suggesting that KP may have a higher diversity of wood-decay fungi. Most of the fungi were observed in more than one park, but some fungi were only detected in one park. For example, R. vinctus, G. webrianum, and F. senex were only detected in ZBG. In this study, the samples were collected from 13 living tree species and dead wood or wood stumps, fallen branches, and shrubs in the four parks. More wood-decay fungal species were detected on dead trees or wood stumps and F. microcarpa than the rest of the wood types (Table 3).

The Colonization of Wood-Decay Fungi on Old Trees and Their Significance
Colonization rate of wood-decay fungi on old trees was higher than that on the younger ones. Of the four parks, 23% of OVTs (large trees in the case of HKO) were infected with wood-decay fungi, and the infection rates were 29%, 16%, 20%, and 35% for HKP, ZBG, KP, and HKO, respectively ( Figure 6A). However, the occurrence rate of wood-decay fungi in all trees in these parks was only around 0.7% ( Figure 6A). More wood-decay fungi were discovered from OVTs than those from younger trees (t = 2.496, df = 3, p = 0.0440). Of all the fungal samples collected, 70% were detected from OVTs (or large trees in the case of HKO), and 30% from younger trees and stumps. The ratio between the numbers of wood-decay fungi samples collected from OVTs and the numbers of wood-decay fungi samples collected from non-OVTs were 7:1, 1:1, 2.8:1, and 2:1 for HKP, ZBG, KP, and HKO, respectively ( Figure 6B).

Discussion
In this study, we surveyed four major parks in Hong Kong for detection of common wood-decay fungi that affected the urban forests in the cosmopolitan Hong Kong. The major wood-decay fungi found in this study were species in Hymenochaetaceae and Polyporales. The family of Hymenochaetaceae contains many species that may cause diseases in broad-leaved and coniferous trees, causing heart rot, canker, and root rot diseases. Phellinus noxius and F. senex from this group were detected in this study and both are known for causing white rot [15]. Other species in this family, such as the Phellinus igniarius group, are usually found as parasites on broad-leaved wood plants, cause white-rot of heartwood [37][38][39][40][41]. The Polyporales were mostly saprophytes and contain a large number of wood-decay fungi, in which a portion of them could be pathogenic [42,43]. Species within this order such as Daedaleopsis confragosa (Bolton) J. Schröt. and Fomes fomentarius (L.) Fr., can cause white trunk rot, and Ganoderma Austral N. Maek., Suhara & R. Kondo and G. webrianum, can cause white-rot and butt-rot [37]. Ganoderma spp., Earliella spp., Hexagonia spp., Ceriporia spp., and Physisporinus spp. Were detected in this study, which were also common wood-decay fungi affects urban trees [18,44,45]. Fungi within Polyporales accounted for 50% of all the wood-decay fungi in this study, suggesting the role as the main wood rotter in the four parks of Hong Kong. This was in line with previous surveys on wood-decay fungi in urban trees in that Polyporales were dominant fungi in urban tree failures [18,44]. The highest diversity of wood-decay fungal community was observed in KP, which could be due to the existence of the largest number of OVTs among the four surveyed parks. Interestingly, ZBG seemed to host a unique group of wood-decay fungi comparing to the other parks. This could be a result of the unique plant community of ZBG that might provide substrates for a different wood-decay fungal community [46].
Plant diseases were traditionally detected with the observation of symptoms and signs. However, some diseases do not present obvious symptoms or fruiting bodies at the early stage of infection. In this case, instead of diagnosing based on observations, molecular techniques could be applied [47]. For the ITS sequences generated in this study, 86% of them could be identified to the species level. The ITS sequences was also commonly used in other wood-decay fungi studies [18,48]. It is applicable in this type of study, but additional genetic regions that are less conservative, such as β-tubulin and elongation factor, could be used for a more accurate speciation [49].
P. noxius contributed to a large percentage of wood-decay fungi in the four parks investigated, especially in HKO. The pathogen is highly virulent in a number of tree species [50,51] and trees may show discoloration in foliage within 2 months and significant decline over periods of one year or more [15]. Trees with observable decline and dieback were more likely to be diagnosed and therefore P. noxius ended up with a high frequency of detection. The main spreading strategy of P. noxius was through root contacts, which enables the pathogen to cause an overwhelming break out of brown-root-rot disease to a large area of densely planted trees [15]. For the same reason, a cluster of infection was observed at the entrance in HKO. On the other hand, the pathogen can also survive as a saprophyte and could remain infectious on the wood debris for up to a decade, making it difficult to eliminate the pathogen once it was established [52]. Therefore, between the cluster of infected A. moluccana at the entrance of HKO and the infected A. moluccana 50 m away inside HKO, a number of stumps, bushes, and trees were found to be infested with P. noxius. If these infected trees were not completely removed and infested soil untreated, more trees in HKO would be infected [53].
Stumps and dead trees were expected to host a large number of wood-decay fungi because these fungi have a role as saprophytic decomposers. Unfortunately, F. microcarpa was also found to host a variety of different fungi. Ficus microcarpa, especially the ones that were identified as OVTs, had widely spread branches and numerous large aerial roots that were exposed to the humid air. There might be a greater chance of microbial colonization on these F. microcarpa than other trees. In addition, the spaces among aerial roots and the main stems may trap water for an extended time, making it a conducive environment for fungal colonization. According to Table 3, P. noxius was detected on five types of host in this study, indicating a potential wide host range, which was in line with previous reports from Taiwan that P. noxius could infect more than 200 tree species [15,51]. The other wood-decay fungi with more than one occurrence were also found in different tree species. Although there was no obvious evidence to prove the relationship between the host trees and specific pathogens in this study, some tree species such as F. microcarpa, were more likely to be colonized by wood-decay fungi in general than others [54].
The severity of rot disease of trees depended on the aggressiveness of wood-decay fungi, susceptibility of the host tree species, as well as the environmental conditions. Old trees could be more vulnerable to decay fungi than young trees under environmental and senescent stresses [55]. Compared to young trees, more old trees were observed with wood-decay fungi but with less significance. The most aggressive pathogenic wood-decay fungus discovered from this study was P. noxius ( Figure S2). The pathogen infected OVTs in HKP, KP, and several large trees in HKO, which were still alive at the time of the second sampling despite the declining leaves and rot at the base of the stem, whereas bushes and smaller trees infected in HKO were already dead. Old and large trees may have extended root systems or plenty of aerial roots, as for F. microcarpa, to support the trees for a certain period of time, while for small trees, the infection of the main stems resulted in the blocking of nutrients and water supplies from the soil.
A variation in disease severity and progression was observed with each fungal species affecting a different tree species. Physisporinus vitreus could also cause root-rot diseases, but the severity was observed to be dependent upon the tree species ( Figure S3). An M. indica tree in HKP infected by P. vitreus had been removed already during the study period because of the severe rot in the roots, while a L. rhodostegia tree in ZBG only showed fruiting bodies of P. vitreus without any obvious symptoms of root-rot. This may be because of host specificity, or due to different stages of infection. Fuscoporia senex is a white-rot fungus which usually causes stem-rot [56]. It had been discovered more on Fabaceae trees in Hong Kong, but fruiting bodies of fungi were also discovered on stumps. Though F. senex was only discovered in ZBG but, for both trees, it caused significant cavities on the tree trunks. Similar cavities were also discovered on other old trees, either on the stem or the base trunk, caused by Ganoderma species, such as G. applanatum, which was found in HKP and KP, laterally attached to the surface of the cavity of the trees, and G. gibbosum, which was found at HKP, KP, and HKO, laterally attached to old trees or old wood stumps ( Figure S4). However, pathogenic fungi, such as the above-mentioned P. noxius, was found in both small and large trees in HKO, presenting high aggressiveness and causing tree decline in a rather short time. In contrast, trees colonized by less aggressive wood-decay fungi such as Ganoderma spp., did not cause quick decline of trees even though cavities formed in the tree trunk. The pathogenicity of a wood-decay fungus was also affected by its environment and hosts, which may cause a shift in their trophic mode. Earliella scabrosa were found in both KP and HKO, but their trophic modes were different. In HKO, E. scabrosa was found on a fallen branch, as a saprobe, while in KP, the fruiting body of E. scabrosa was found laterally attached to a F. microcarpa YTM/65, as a rot pathogen ( Figure S3).
Though for most of the trees, only one wood-decay fungal species was detected, it was possible that one tree could be simultaneously colonized by two or more wood-decay fungal species [39]. The F. microcarpa YTM/65, located in KP, saw both E. scabrosa and P. noxius detected. It was assumed that P. noxius could result in a more serious problem for the tree since the decayed roots were already penetrated by mycelia of P. noxius. Another case was the F. microcarpa CW/110 in HKP, of which the root was mostly colonized by P. noxius, while the cavity of the base trunk was more likely to be caused by G. gibbosum. The interactions between the two parasites on a single tree were unclear, but they may collectively facilitate the wood-decay process, or they may not interact at all due to their colonization on different parts of the trees.
Urban trees are valuable in many ways and a better understanding of common wood-decay fungi in an area facilitates the protection and management of trees against rot and risk. To better protect the trees, regular inspection should be conducted for proactive management [23]. Early detection of fungal infection makes it possible to save the tree by removing infected tissues and to replace highly hazardous trees gradually [5]. HKP, ZBG, and KP receive a large number of visitors daily, so weak trees can be dangerous if not identified and removed in a timely manner. When it is necessary to remove a diseased tree, it is crucial to clean up the infected area for replanting trees, especially if it is a root rot fungus. Trees should be managed by following an appropriate protocol. Pruning should be avoided during fungal sporulation seasons and pruning wounds should be protected to avoid wood-decay fungi infections [18].

Conclusions
This survey, emphasizing the urbanized ecosystem by including trees from four public parks, provided a basis for the baseline determination of fungal infection, conservation, protection, and management of urban trees. In this study, 28 wood-decay fungal species were detected in HKP, ZBG, KP and HKO. Among them P. noxius, G. gibbosum, and A. polytricha were the most abundant species and occurred in three of the four parks. Across the tree species, F. microcarpa were most vulnerable to wood-decay fungi with different species, while dead wood and wood stumps were more likely to be colonized by saprotrophic wood-decay fungi. The colonization rate of wood-decay fungi on older trees was much higher than that on the younger ones, resulting in infections and the subsequent problems were more likely shown in the older trees.