Geographic locality greatly influences fungal endophyte communities in Cephalotaxus harringtonia

Although endophytes of conifers have been extensively studied, few data are available on Cephalotaxaceae. We examined foliar and stem endophytes of Cephalotaxus harringtonia, within its natural range in Japan and outside its natural range in France to study the effect of geography on endophyte community composition. In Japan, rapidly growing endophytes were dominant and may have masked the real diversity, in comparison to France where most endophytes were growing slowly. Analyses of ITS rDNA revealed 104 different Blast Groups among 554 isolates. Almost no overlap between endophyte assemblages of C. harringtonia from the two countries was observed. It seems that Japanese C. harringtonia trees, which should be well adapted to their native site, would host a specific, endemic endophyte community, while trees that have been introduced recently to a foreign site, in France, should have captured existing cosmopolitan and more generalist taxa. In Japan the majority of xylariaceous taxa, which dominated the communities, were unknown and, although closely related to Asian taxa, may be new to science. Dothideomycetes were more prevalent in France. Locally, urban environment, particularly in Japan, may have introduced some perturbations in the native endophyte community of C. harringtonia, with an abundance of generalist fungi such as Nigrospora and Colletotrichum.     

Non-target impacts of soybean rust fungicides on the fungal entomopathogens of soybean aphid

Soybean aphid, Aphis glycines, has caused serious economic damage to soybean across the North Central US since its introduction to North America in 2000. The management of another invasive soybean pest, Asian soybean rust, Phakopsora pachyrhizi, using foliar fungicide applications has the potential to impact soybean aphid populations by suppressing beneficial fungal entomopathogens. In 2005 and 2006, we applied recommended soybean rust fungicide treatments, consisting of strobilurin and triazole fungicides, to small soybean plots in two locations to assess if such applications might suppress aphid fungal epizootics. In Lamberton, MN, in 2005, during the epizootic, fungicide-treated plots averaged 2.0 ± 0.7% (mean ± SE) disease prevalence while untreated plots averaged 14.2 ± 5.6%. In 2007, we applied strobilurin and strobilurin-triazole mix fungicides to single-plant microplots either before or after release of Pandora neoaphidis, the most commonly observed aphid pathogen in 2005 and 2006. Treatments that contained a mixture of two active ingredients significantly lowered peak and cumulative aphid disease prevalence in both early and late reproductive stage soybeans indicating that fungicide mixtures used to manage soybean rust can negatively impact an aphid-specific fungal pathogen. However, no consistent soybean aphid population response was observed in these studies of low levels of aphid fungal infection.     

Fungal endophytes from Acer ginnala Maxim: isolation, identification and their yield of gallic acid

Aims:  The aim of the study was to isolate the endophytic fungi from Acer ginnala and screen isolates rich in gallic acid.
Methods and Results:  After epiphytic sterilization, 145 fungal endophytes were isolated from the stem, annual twig and seed of Acer ginnala . The endophytes were grouped into ten different taxa, Phomopsis sp., Neurospora sp., Phoma sp., Epicoccum sp., Penicillium sp., Alternaria sp., Fusarium sp., Trichoderma sp., Cladosporium sp. and a species of Pleosporales Incertae Sedis , by their morphological traits and ITS-rDNA sequence analysis. The content and yield of gallic acid of 141 isolates were determined by HPLC. On average, the species of Pleosporales Incertae Sedis had the highest content and yield of gallic acid (13·28 mg g⁻¹ DW; 119·62 mg l⁻¹), while Alternaria sp. had the lowest.
Conclusions:  Of 141 fungal endophytes from A. ginnala , Phomopsis sp. isolate SX10 showed both the highest content and the highest yield of gallic acid (29·25 mg g⁻¹ DW; 200·47 mg l⁻¹).
Significance and Impact of the Study:  Endophytic fungi isolated from A. ginnala may be used as potential producers of gallic acid and other compounds with biological activities, or functioned as elicitors to produce natural compounds.     

The community of needle endophytes reflects the current physiological state of Norway spruce

This study investigated fungal endophytes in the needles of Norway spruce (Picea abies) cuttings in relation to host tree growth. We also determined the prevalence of endophytes in needles incubated for six months. The cuttings originated from clonal origins showing slow- and fast-growth in long-term field trials but the heritable differences in growth rate were not yet detected among the studied cutting. Endophytes were isolated from surface-sterilized needles with culture-free DNA techniques. No significant differences were observed between endophyte communities of slow- and fast-growing clonal origins. However, the endophyte community correlated with the current growth rate of cuttings suggesting that endophytes reflect short- rather than long-term performance of a host. The concentration of condensed tannins was similar in slow- and fast-growing clonal origins but it showed a negative relationship with endophyte species richness, implying that these secondary compounds may play an important role in spruce tolerance against fungal infections. More than a third of endophyte species were detected in both fresh and decomposing needles, indicating that many needle endophytes are facultative saprotrophs. Several potentially pathogenic fungal species were also found within the community of saprotrophic endophytes.     

High diversity of endophytic fungi from the pharmaceutical plant, Heterosmilax japonica Kunth revealed by cultivation-independent approach

Heterosmilax japonica Kunth is well recognized for its diuretic effects in China. However, little is known about its endophytic fungi. In this study, microbial communities inhabiting the stems of H. japonica in spring and summer were investigated by light microscopy and cultivation-independent approaches, such as RFLP analysis and sequencing of rDNA ITS library. Molecular phylogenetic analysis showed that a broad spectrum of fungi, including Mycosphaerella, Phomopsis, Aureobasidium, Cladosporium, Glomerella, Botryosphaeria, Guignardia, is able to colonize the plants internally. Particularly, several rDNA sequences determined in this study like YJ4-61 are not specifically affiliated with any currently documented fungal sequences in the public database. Several sequence types, such as YJ4-9 and YJ4-70, are significantly similar to some uncultured environmental samples. Furthermore, our result also showed that the samples collected in spring harbored more abundant endophytic populations than that in summer, implying a seasonal fluctuation for the endophytes in H. japonica.     

内生真菌对羽茅生长及光合特性的影响

通过温室栽培实验,以感染两种内生真菌(Neotyphodium sibiricum和Neotyphodium gansuence)和未感染内生真菌的羽茅(Achnatherum sibiricum)为实验材料,分析感染不同种内生真菌对宿主植物的生长及光合特性的影响。结果表明,感染两种内生真菌羽茅的株高和CO₂补偿点显著低于未染菌的羽茅,而染菌羽茅的蒸腾速率和气孔导度显著高于未染菌羽茅,但对于感染不同种内生真菌的羽茅,无论是分蘖数与生物量的积累还是光合生理值之间均无显著差异。     

A novel plant-fungal mutualism associated with fire

Bromus tectorum, or cheatgrass, is native to Eurasia and widely invasive in western North America. By late spring, this annual plant has dispersed its seed and died; its aboveground biomass then becomes fine fuel that burns as frequently as once every 3-5 y in its invaded range. Cheatgrass has proven to be better adapted to fire there than many competing plants, but the contribution of its fungal symbionts to this adaptation had not previously been studied. In sampling cheatgrass endophytes, many fire-associated fungi were found, including Morchella in three western states (New Mexico, Idaho, and Washington). In greenhouse experiments, a New Mexico isolate of Morchella increased both the biomass and fecundity of its local cheatgrass population, thus simultaneously increasing both the probability of fire and survival of that event, via more fuel and a greater, belowground seed bank, respectively. Re-isolation efforts proved that Morchella could infect cheatgrass roots in a non-mycorrhizal manner and then grow up into aboveground tissues. The same Morchella isolate also increased survival of seed exposed to heat typical of that which develops in the seed bank during a cheatgrass fire. Phylogenetic analysis of Eurasian and North American Morchella revealed that this fire-associated mutualism was evolutionarily novel, in that cheatgrass isolates belonged to two phylogenetically distinct species, or phylotypes, designated Mel-6 and Mel-12 whose evolutionary origin appears to be within western North America. Mutualisms with fire-associated fungi may be contributing to the cheatgrass invasion of western North America.     

Chemical and molecular characterization of Phomopsis and Cytospora-like endophytes from different host plants in Brazil

Phomopsis and related taxa comprise important endophytic and plant pathogenic species, and are known for the production of a diverse array of secondary metabolites. Species concepts within this group based on morphological characters and assumed host specificity do not reflect phylogenetic affinities. Additional phenotypic characters, such as profiles of secondary metabolites, are needed for practical species recognition. We investigated 36 strains of Phomopsis spp. and Cytospora-like fungi, obtained as endophytes of different host plants in Brazil, using metabolite profiling based on HPLC-UV/liquid chromatography -mass spectrometry (LC-MS) combined with cluster analysis of the results. Strains were also subjected to phylogenetic analyses based on internal transcribed spacer (ITS) rDNA. Six chemotypes were identified. Chemotypes 1-5 contained Phomopsis strains, while Cytospora-like strains formed the chemotype 6. Strains of chemotype 1 typically produced alternariols, altenusin, altenuene, cytosporones, and dothiorelones. Alternariol and seven unknown compounds were consistently produced by strains of chemotype 2. Members of chemotypes 3-5 produced poor metabolite profiles containing few chemical markers. Cytospora-like endophytes (chemotype 6) produced a characteristic set of metabolites including cytosporones and dothiorelones. Bayesian and Maximum Parsimony (MP) trees classified strains of each chemotype into single phylogenetic lineages or closely related groups. Strains of chemotypes 1 and 2 formed a monophyletic group along with Diaporthe neotheicola. The remaining Phomopsis strains formed monophyletic (chemotype 4) or polyphyletic (chemotypes 3 and 5) lineages inside a large and well supported clade. Cytospora-like strains formed a monophyletic lineage located at an intermediary position between Diaporthe/Phomopsis and Valsa/Cytospora clades. The combined results show that the production of secondary metabolites by Phomopsis and related Diaporthales may be species-specific, giving support to the use of metabolite profiling and chemical classification for phenotypic recognition and delimitation of species.     

Fungal endophytes in seeds and needles of Pinus monticola

Using a sequence-based approach, we investigated the transmission of diverse fungal endophytes in seed and needles of Pinus monticola, western white pine. We isolated 2003 fungal endophytes from 750 surface-sterilized needles. In contrast, only 16 endophytic isolates were obtained from 800 surface-sterilized seeds. The ITS region was sequenced from a representative selection of these endophytes. Isolates were then assigned to the most closely related taxa in GenBank. Although 95 % of the endophytes in needles from mature trees belonged to the Rhytismataceae, 82 unique ITS sequences were obtained from at least 21 genera and 10 different orders of fungi. Significantly, none of the endophytes in seed were rhytismataceous (χ² = 180; P < 0.001). Similarly, needles of greenhouse seedlings yielded only non-rhytismataceous isolates, whereas seedlings of the same age that had naturally regenerated near older white pines in roadless areas were colonized by rhytismataceous endophytes almost to the same extent as in mature trees. Only one of 17 rhytismataceous isolates were able to grow on a medium containing only 0.17 % nitrogen, whereas 25 of 31 non-rhytismataceous endophytes grew. Rhytismataceous endophytes are dominant in needles of P. monticola, but they appear to be absent in seed, and unlikely colonists of nitrogen-limiting host tissues such as the apoplast.     

Wheat cells accumulate a syringyl-rich lignin during the hypersensitive resistance response

The stem rust fungus Puccinia graminis f.sp. tritici is an obligately biotrophic pathogen attacking wheat (Triticum aestivum). In compatible host/pathogen-interactions, the fungus participates in the host's metabolism by establishing functional haustoria in the susceptible plant cells. In highly resistant wheat cultivars, fungal attack is stopped by a hypersensitive response of penetrated host cells. This mechanism of programmed cell death of single plant cells is accompanied by the intracellular accumulation of material with UV-fluorescence typical of phenolic compounds. A similar reaction can be induced in healthy wheat leaves by the application of a rust-derived elicitor. We analysed the biochemical composition of this defense-induced phenolic material. Contents of total soluble and cell wall esterified and etherified phenolic acids were determined in rust-inoculated and elicitor-treated leaves of the fully susceptible wheat cultivar Prelude and its highly resistant, near-isogenic line Prelude-Sr5. While no resistance-related changes occured in any of these fractions, the lignin content as determined by the thioglycolic acid and the acetyl bromide methods increased after elicitor treatment. Nitrobenzene oxidation revealed that the entire increase can be explained by an increase in syringyl units only. These biochemical data were confirmed by fluorescence emission spectra analyses which indicated a defense-induced enrichment of syringyl lignin for cell wall samples both from elicitor-treated wheat leaves and single host cells undergoing a hypersensitive response upon fungal penetration.     

内生真菌感染对宿主羊草抗病性的影响

以感染内生真菌的天然禾草羊草为实验材料,通过体外纯培养条件下的内生真菌、感染内生真菌的离体叶片和在体叶片对3种病原菌的抑菌实验,以探讨内生真菌对宿主植物羊草在抗病性方面的贡献。结果表明:体外纯培养条件下,分离自羊草的内生真菌Epichlobromicola对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)这3种病原菌都具有抑制作用,抑菌率分别达56.22%,46.93%和45.15%,且内生真菌培养滤液可以有效抑制这3种病原菌的孢子萌发,平均萌发率分别为30.4%,15.7%和16.4%;宿主植物叶片在离体条件下,内生真菌感染可以有效降低羊草叶片受C.lunata和C.sp.侵染后的病斑数或病斑长度,但对B.sorokiniana不起作用,甚至提高了叶片的病斑数及病斑长度,而离体叶片提取液对不同病原菌均有不同程度的抑制作用;在体条件下,内生真菌均可以通过降低叶片病斑数来增强羊草植株对这3种病原菌的抗性。由此看来,内生真菌E.bromicola对宿主植物羊草在抗病原菌侵染方面有一定的增益作用。     

Contingency rules for pathogen competition and antagonism in a genetically based,plant defense hierarchy

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Antioxidant and anticancer activity of extract from Betula platyphylla var. japonica

The antioxidant and anticancer properties of a medicinal plant, Betula platyphylla var. japonica were investigated. The total methanol extract of B. platyphylla var. japonica had protective effects against hydrogen peroxide (H2O2) in the Chinese hamster lung fibroblast (V79-4) cell line and induced apoptotic cell death in human promyelocytic leukemia (HL-60) cells, a cancer cell line. B. platyphylla var. japonica extract significantly increased cell viability against H2O2. The extract also showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 2.4 microg/ml) and lipid peroxidation inhibitory activity (IC50 below 4.0 microg/ml). Furthermore, B. platyphylla var. japonica extract reduced the number of V79-4 cells arrested in G2/M in response to H2O2 treatment and increased the activities of several cellular antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. Treatment with B. platyphylla var. japonica extract induced cytotoxicity and apoptosis in HL-60 cells, as shown by nucleosomal DNA fragmentation, increases in the subdiploid cell population, and fluorescence microscopy. B. platyphylla var. japonica extract gradually increased the expression of pro-apoptotic Bax and led to the activation of caspase-3 and cleavage of PARP. These findings suggest that B. platyphylla var. japonica exhibits potential antioxidant and anticancer properties.     

Naphthoquinone spiroketal with allelochemical activity from the newly discovered endophytic fungus Edenia gomezpompae

Chemical investigation of the mycelium of Edenia gomezpompae, a newly discovered endophytic fungus isolated from the leaves of Callicarpa acuminata (Verbenaceae) collected from the ecological reserve El Eden, Quintana Roo, Mexico, resulted in the isolation of four naphthoquinone spiroketals, including three new compounds and palmarumycin CP2 (4). We elucidated the structures of the metabolites by extensive NMR spectroscopy studies, including DEPT, COSY, NOESY, HSQC, HMBC, and chiroptical methods. The trivial names proposed for these compounds are preussomerin EG1 (1), preussomerin EG2 (2) and preussomerin EG3 (3). In addition, the X-ray data for 4 were obtained. The bioactivity of the mycelial organic extracts and the pure compounds was tested against three endophytic fungi (Colletotrichum sp., Phomopsis sp., and Guignardia manguifera) isolated from the same plant species (C. acuminata, Verbenaceae) and against four economically important phytopathogenic microorganisms (two fungoid oomycetes, Phythophtora capsici and Phythophtora parasitica, and the fungi Fusarium oxysporum and Alternaria solani). Spiroketals 1-3 displayed significant growth inhibition against all the phytopathogens. IC50 values for the four phytopathogens were from 20 to 170 microg/ml. Palmarumycin CP2 (4) was not bioactive against any of the fungi tested. Compound 1 showed the strongest bioactivity. The acetylated derivatives of preussomerin EG1 (1), 1a and 1b, were obtained and their biological activity was tested on endophytes and phytopathogens. Preussomerin EG1 1, 1a and 1b exhibited significant bioactivity against all microorganisms tested with the exception of Alternaria solani. This is the first report of allelochemicals with antifungal activity from the newly discovered endophytic fungus E. gomezpompae.     

Fungal endophytes from the three-leaved caper,Crataeva magna (Lour.) DC. (Capparidaceae)

Fungal endophytes were isolated from Crataeva magna, a medicinal plant growing along the streams and rivers, constituting riparian vegetation in Karnataka, southern India. Fresh bark and twig pieces were used for the isolation using standard methods. Ninety-six endophytic fungal isolates were isolated from 800 bark and twig segments. Mitosporic fungi represented as a major group (85%) followed by zygomycetes (10%) and ascomycetes (5%). Bark samples contained more endophytes than twig samples. Verticillium, Nigrospora oryzae and Fusarium verticilloides were the dominant fungal endophytes.     

Chemical profiling of Ocimum americanum using external flavonoids

A HPLC survey was undertaken of the external flavonoids in 111 herbarium specimens of Ocimum americanum L. (O. canum Sims), which were largely collected from their natural habitats throughout Africa and Asia. The purpose of this study was to establish the flavonoid profiles of this species over the full range of its geographic distribution in order to use these for authentication purposes. Six different external flavonoid chemotypes were found. The major chemotype, present in circa 80% of the specimens of both var. americanum and var. pilosum collected throughout the distribution area of the species, was characterised by very high levels of nevadensin, slightly lower levels of salvigenin and much lower levels of up to 15 other external flavones. Of the remaining five chemotypes, two were found in var. americanum and three in var. pilosum. All specimens belonging to these chemotypes were collected in South or East Africa and represented by only a few specimens. These samples contained much smaller levels of flavones than present in the major chemotype of O. americanum and all lacked nevadensin. Xanthomicrol, a compound absent from the main chemotype, was the dominant flavone in two of the minor chemotypes. The external flavonoid profiles found in the six chemotypes of O. americanum were compared with those of O. x citriodorum (11 herbarium specimens studied) and seven other closely related species of Ocimum. The main nevadensin/salvigenin pattern present in O. americanum was also found in O. x citriodorum, O. basilicum and some specimens of O. minimum, but there were strong quantitative differences in external flavonoids among these taxa. The other chemotypes of O. americanum showed some similarities in their external flavone profiles to those found in the closely related East African species O. fischeri, O. forskolei, O. kenyense and O. kilimandscharicum, which occur in the same geographic areas. This suggests that the uncommon chemotypes of O. americanum may have originated by an exchange of genes with other Ocimum species, e.g. by introgressive hybridisation. Despite some similarities in profiles, chemical differences were also found among the species, so that it should be possible to authenticate a large proportion of leaf samples of O. americanum on the basis of external flavonoid profiles.     

Drazepinone, a trisubstituted tetrahydronaphthofuroazepinone with herbicidal activity produced by Drechslera siccans

When grown in a minimal-defined medium, a strain of Drechslera siccans, a pathogenic fungus isolated from seeds of Lolium perenne, produced phytotoxic metabolites. This strain is one of the best toxin producers among several grass pathogenic fungal strains collected and tested to find phytotoxins to be used as natural herbicides of monocot weeds. From the culture filtrates of D. siccans, we isolated a new phytotoxic trisubstituted naphthofuroazepinone, named drazepinone, and characterised it as a 3,5,12a-trimethyl-2,5,5a,12a-tetrahydro-1H-naphtho[2',3':4,5]furo[2,3-b]azepin-2-one. Assayed at 2 microg microl(-1) solution the novel metabolite proved to have broad-spectrum herbicidal properties, without antibacterial and antifungal activities, and low zootoxic activity. Its original chemical structure and the interesting biological properties make drazepinone a potential natural herbicide.     

Peramine and other fungal alkaloids are exuded in the guttation fluid of endophyte-infected grasses

Many grasses live in association with asymptomatic fungi (Neotyphodium spp. endophytes), which grow in the intercellular spaces of the grass. These endophytes produce a range of alkaloids that protect the grass against grazing by mammals and insects. One of these alkaloids is an unusual pyrrolopyrazine, peramine. Peramine appears to be continuously produced by the endophyte, but does not progressively accumulate. No mechanism for the removal of peramine by its further metabolism or any other process has been reported. Our aim was to detect peramine or peramine metabolites in plant fluids to determine if peramine is mobilized, metabolized or excreted by the plant. We also wanted to determine if other fungal metabolites are mobilized by the plant, as has been proposed for the loline alkaloids.We developed a highly sensitive method for the analysis of peramine, using a linear ion trap mass spectrometer. We studied the fragmentation pathway of peramine using ESI MSⁿ and ESI FTICRMS. Based on these results we developed a single reaction monitoring method using the fragmentation of the guanidinium moiety. Cut leaf fluid and guttation fluid of different grass endophyte associations (Lolium perenne with Neotyphodium lolii, Festuca arundinacea with Neotyphodium coenophialum, and Elymus sp. with Epichloë sp.) were analysed. Peramine was detected in the cut leaf fluid of all grass-endophyte associations, but not in the guttation fluid of all associations. In some associations we also detected lolines and ergot peptide alkaloids. This is the first report showing the mobilization of fungal alkaloids into plant fluids by the host plant in grass-endophyte associations.     

福州大叶榕隐头果内的小蜂群落结构与多样性

对福州2个样地10株大叶榕果内小蜂群落组成和物种多样性进行研究。全年在两个样地267个隐头果内共收集到小蜂13458只。发现大叶榕隐头果内有7种小蜂,隶属小蜂总科Chalcidoidae中的榕小蜂科Agaonidae、金小蜂科Pteromalidae的隐针榕小蜂亚科Epichrysomallinae、锥尾榕小蜂亚科Otitesellinae和延腹榕小蜂亚科Sycoryctinae;广肩小蜂科Eurytomidae、刻腹小蜂科Ormyridae、姬小蜂科Eulophidae,其中榕小蜂科的Platyscapa coronata是大叶榕唯一的传粉者,传粉方式为主动传粉;非传粉榕小蜂的雄性多型现象普遍。传粉与非传粉小蜂的性比明显具有偏雌现象。传粉小蜂性比为0.19±0.07,非传粉小蜂中Camarothorax bismasculinus小蜂性比为0.36±0.10;Walkerella sp.小蜂性比为0.36±0.22,Sycoscapter sp.小蜂性比为0.31±0.22,Sycophila sp.小蜂性比为0.35±0.13。雄性多型现象可能是导致非传粉小蜂性比提高的原因之一。根据各种榕小蜂发生数量及连续性,可将大叶榕隐头果中的榕小蜂分为常见种和偶见种,Platyscapa coronata、Camarothorax bismasculinus、Walkerella sp.、Sycoscapter sp.和Sycophila sp.为常见种,Omyrus sp.和Aprostocetus sp.为偶见种。偶见种的存在对常见种的数量几乎没有影响,偶见种利用的是榕果内未饱和的一部分资源,也可能是榕果为偶见种的发生预留了空间和资源。首次发现姬小蜂科的昆虫寄生在榕果内,且有一定的种群数量,为姬小蜂科昆虫分类及其生物学、生态学特性研究提供了基础资料。在大叶榕小蜂群落结构中,传粉小蜂和非传粉小蜂的种类和数量呈现明显的季节性。冬-春季(12-翌年5月)榕果内小蜂的种类和数量较多,传粉榕小蜂是优势种,其重要值达到0.42,榕果种子结实率高;夏-秋季(6-11月间)小蜂种类和数量略少,Camarothorax bismasculinus和Sycophila sp.是优势种,其重要值分别为0.56和0.28,而传粉小蜂的重要值仅为0.025,此期大叶榕榕果中几乎找不到传粉小蜂,榕果结实率极低,对大叶榕的繁殖利益有较大的负面影响。雨水和高温等不良气候,可能是导致夏-秋季雄花期榕果内的传粉小蜂数量骤减的主要原因。两个实验样地的小蜂群落结构组成没有明显差异,年变化趋势相似。研究结果为城市绿化和热带雨林生物多样性保护提供科学依据。