Diversity and bioactivity of bacterial endophyte community of Cupressaceae

The plant family Cupressaceae (Coniferales) harbours diverse endophytic fungi with antifungal, antibacterial and antiproliferative activities. Here, endophytic association of a broad bacterial community with the healthy foliar tissues of Cupressus arizonica, Cupressus sempervirens, Juniperus communis and Thuja orientalis (Cupressaceae) is shown. We isolated over 69 endophytic bacterial strains of Proteobacteria, Bacilli and Actinobacteria from Cupressaceae. The initial screening for antifungal activity against Pyricularia oryzae identified eleven superior bacterial strains which were identified as Brevundimonas diminuta CAE₂₄ (G^?, Caulobacteraceae, Alphaproteobacteria); Stenotrophomonas maltophilia CAE₂₃, CSE₄, CSE₁₂, CSE₄₂, CSE₄₉ and CSE₆₂ (G^?, Xanthomonadaceae, Gammaproteobacteria); Bacillus pumilus CSE₆₆ and Bacillus subtilis POE₂₆ (G⁺, Bacillaceae, Bacilli); and Microbacterium resistens CSE₁₉ (G⁺, Microbacteriaceae, Actinobacteria). The dominant isolated bacterial species was S. maltophilia. This species represented 63.6% of the superior strains which was ubiquitous and also non‐host specific. In addition, the superior bacterial strains produced bioactive secondary metabolites and volatile compounds (VOCs) with antifungal activity against the fungal pathogens of Cupressaceae, that is Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. The antagonistic activity of the endophytic bacteria on the target fungi was also confirmed in vitro. To our knowledge, this is the first to document such an endophytic bacterial community in Cupressaceae and its bioactivity. These findings may find application in organic agroforestry for plant disease biocontrol and in biopharmacy for lead molecule discovery.     

Assessing carbon sequestration in plantation forests of important conifers based on the system of permanent sample plots across Taiwan

ABSTRACT

Plantation forests play a critical role in forest management due to their high productivity and large contribution to carbon sequestration (CSE). The purpose of this study was to assess the CSE of plantations containing four important conifer species distributed across Taiwan, namely, the China fir (Cunninghamia lanceolata), Japanese cedar (Cryptomeria japonica), Taiwania (Taiwania cryptomerioides) and Taiwan red cypress (Chamaecyparis formosensis). Data regarding the plantations were obtained from a survey of permanent sample plots (PSPs). We used these data to calculate the CSE in each PSP and adopted CSE_mean and CSE_period as indicators to assess the CSE of the four conifers. According to the CSE_mean obtained from analysis of variance and the least significant difference method, two groups were identified among these four conifers: the Japanese cedar (4.03 Mg ha^?1 yr^?1) and Taiwania (3.52 Mg ha^?1 yr^?1) yielded higher CSE_mean values and the China fir (1.79 Mg ha^?1 yr^?1) and Taiwan red cypress (2.36 Mg ha^?1 yr^?1) yielded lower CSE_mean values. The same patterns were observed in the CSE_period values; however, no significant difference in CSE_period was observed between Taiwan red cypress and either of the two groups. Therefore, Japanese cedar and Taiwania have high CSE potential among conifers.     

Pathogenic fungi associated with pre‐ and post‐emergence seedling blight of pine and cypress in Fars Province,Iran

Seed and root rot of pine and cypress seedlings cause heavy annual losses to forest nurseries in Fars Province. Root and crown samples of various species of conifers, such as Tehran pine (Pinus eldarica), brutia pine (Pinus brutia), Arizona cypress (Cupressus arizonica), Shirazian cypress (Cupressus sempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) and oriental arborvitae (Thuja orientalis), showing chlorosis, necrosis, stunted growth, defoliation and root and crown rot symptoms were collected from pine and cypress nurseries across Fars province at various time intervals. Infected tissues were washed and cultured on acidified potato dextrose agar (PDA) and corn meal agar (CMA) amended with Delvocide and ampicillin, with and without surface sterilization, respectively, and incubated at 25°C for 3–5 days. Decaying seeds of all plant species were also collected from nursery seed stocks and cultured on PDA. Hyphal tip isolates were used for further studies. Species of Phytophthora, Pythium, Rhizoctonia and Fusarium were isolated from symptomatic seedlings of pine and cypress at different times during the growing season. Pathogenicity of isolates of Phytophthora, Pythium and Rhizoctonia was confirmed on seedlings of all plant species, whereas Fusarium sp. isolated from the seeds of Tehran pine was only pathogenic on seedlings of Tehran pine and Arizona cypress. Virulent isolates identified as Phytophthora nicotiana var. parasitica, Pythium ultimum, Pythium paroecandrum, Rhizoctonia solani and binucleate Rhizoctonia‐like fungus caused root and crown rot of pine and cypress seedlings. Pathogenic isolates of Fusarium identified as Fusarium proliferatum also caused pre‐emergence seed decay of pine and cypress. Isolates of Phytophthora and Pythium were the most virulent of these fungal isolates.     

Sphaeropsis canker,a new disease of cypress in Greece

The fungus Sphaeropsis sp. is reported for first time in Greece to cause cankers on Cupressus sempervirens. The cultural characteristics on PDA, spore shape and size and canker morphology were identical to those of the fungus described as Diplodia pinea f. sp. cupressi in Israel. The cankers of Sphaeropsis sp. are characterized by resin exudation, with fissuring of the bark over a dry sector of the wood. The pathogenicity of the Sphaeropsis sp. was proved by artificial inoculations on selected cypress clones resistant and susceptible to Seiridium cardinale. The mean canker length of the susceptible clone was significantly more than that of the resistant clones. It appears that the defence reaction in cypress against both fungi is similar. In artificial inoculations on Pinus halepensis and Pinus pinea, Sphaeropsis sp. was nonpathogenic. During a 3‐year period, in an experimental plot in western Peloponnese, the fungus spread from one, initially, to 13 cypress clones. Drought stress during the summer appears to be the main factor predisposing the cypress plants to become susceptible to the fungus.     

Molecular evidence of the hybrid origins of leyland cypress (×Cupressocyparis leylandii)

Leyland cypress (×Cupressocyparis leylandii) has been regarded as an intergeneric hybrid between Monterey cypress (Cupressus macrocarpa) and Alaska cypress (Chamaecyparis nootkatensis) in the Cupressaceae. Each partial sequence of the 18S-rDNA from nuclear and therbcL from chloroplast DNA genomes was determined using above three species. At the 59th site from the 5′ end of the 152 bp of the determined 18S-rDNA sequence, one base difference was identified between Monterey cypress (T) and Alaska cypress (A). However, Leyland cypress showed either nucleotide A or T at this site. Since nuclear DNA is inherited by biparental mode, this result shows genetic evidence that the nucleic genome of Leyland cypress originates from Monterey cypress and Alaska cypress and that this species is of hybrid origin.     

Trichoderma viride on cypress shoots and antagonistic action against Seiridium cardinale

The recovery of Trichoderma viride from shoots of Cupressus sempervirens L. in different localities of Northern Italy and its antagonistic action against Seiridium cardinale, agent of the cypress canker, are described.     

‘Le Crete 1’ and ‘Le Crete 2’: two newly patented Seiridium cardinale canker‐resistant cultivars of Cupressus sempervirens

In the Mediterranean area, common cypress (Cupressus sempervirens) has traditionally been used as a multipurpose tree, for its symbolic and ornamental role, for its valuable timber, as well as for windbreaks and soil protection. The epidemic spread of the Seiridium cardinale canker has limited the use of this tree since the 1970s, inducing researchers to develop a breeding programme of cypress aimed at selecting canker‐resistant lines for different uses and to support a flourishing trade of cypress plants. ‘Le Crete 1’ and ‘Le Crete 2’ described here are two new canker‐resistant C. sempervirens varieties patented in 2010, selected through a 13‐year assessment of their response to artificial inoculations and growth traits. Both are characterized by a rapid growth and by a columnar and fastigiated habit that confers them a notable ornamental effect. Preliminary observations showed also that both tend to produce yearly few microsporophylls and little pollen. ‘Le Crete 2’ was also selected for the high growth rate it maintained on heavy, clayey soils.     

Fusarium canker of Italian cypress

A new disease, causing mortality of Italian cypress (Cupressus sempervirens) trees cv. ‘Totem', was observed in an Israeli nursery. Fusarium compactum was isolated from cankers, and its pathogenicity was confirmed by Koch's test. The fungus readily formed conidia and chlamydospores in PDA culture. Growth of F. compactum was fastest at 26–30°C. Artificial inoculations with F. compactum caused severe cankering of several species of each of the following coniferous genera: Cupressus, Juniperus, Tetraclinis, Thuja, Cedrus and Pinus. Canker development was significantly enhanced when extreme water stress was imposed on cypress saplings prior to inoculation.     

Seiridium cardinale cankers in a tolerant Cupressus sempervirens clone under naturally CO2‐enriched conditions

Ramets of the Cupressus sempervirens clone Bolgheri were raised in three CO₂ concentrations in a naturally CO₂‐enriched environment [360 (control), 680 and 1670 μmol mol^–1 CO₂]. Bolgheri is patented as resistant to Seiridium cardinale. The ramets were artificially inoculated using a virulent isolate of S. cardinale. Healing of the resulting cankers was visually assessed over 6 years. Six months after inoculation, cankers on trees grown at 680 μmol mol^–1 were significantly larger than the controls. However, no significant trend was recorded at later dates. In conclusion, the Bolgheri clone maintained its resistance to S. cardinale and could be used to control cypress canker even in increasing atmospheric CO₂ concentrations.     

Fungal endophytes associated with Sonneratia (Sonneratiaceae) mangrove plants on the south coast of China

The aim of this study was to describe endophytic fungal communities residing in five mangrove species (Sonneratia caseolaris, Sonneratia hainanensis, Sonneratia ovata, Sonneratia Paracaseolaris, Sonneratia apetala) collected at the south coast of China. The colonization frequency (CF) of endophytes in roots, stems and leaves was measured and the structures of endophytic communities were investigated, using endophytic fungal diversity indices and similarity coefficients. We found high biodiversity and tissue specificity of endophytic fungi. A total of 391 fungal isolates were identified using morphological and molecular methods. We recovered a total of 39 distinct endophytic species, of which Cytospora, Diaporthe, Fusarium, Glomerella, Mycosphaerella, Phoma, Phomopsis and Stemphylium were the dominant fungal taxa. Most recovered species were found on more than one host species but the dominant species differed according to host and tissue type.     

Phillyrin produced by Colletotrichum gloeosporioides, an endophytic fungus isolated from Forsythia suspensa

To investigate the phillyrin-producing endophytic fungi from the medicinal plant Forsythia suspensa, a total of 24 strains of endophytic fungi were isolated from the healthy stems, leaves and fruits of the plant, 9 from stems, 5 from leaves, and 10 from fruits respectively. All fungal isolates were fermented in liquid PDA medium and their extracts were preliminary analyzed by TLC. One isolated strain G10, which was from the fruit of F. suspensa, had the same R_f value as authentic phillyrin. The potential phillyrin-producing fungus G10 was further analyzed by HPLC and HPLC-MS, and the results showed that the isolate G10 possessed of a retention time and ion peaks identical with the authentic compound phillyrin. The isolate G10 was identified as Colletotrichum gloeosporioides based on the morphological characteristics and ITS sequence analysis. The current research indicates that the numerous endophytic fungi inside the medicinal plants are precious resource for the pharmaceutical natural products that are originally from the plants.     

Genetic differences between autochthonous and breeding populations of common cypress (Cupressus sempervirens L.) in Greece

Individuals were selected for resistance against the imperfect fungus Seiridium cardinale Wag in six common cypress populations (Cupressus sempervirens L.) in Greece. A total of 109 resistant clones and 140 trees from their base populations were surveyed at several allozyme loci. The comparison yielded information on changes in genetic variation due to artificial selection. The genetic structure of the breeding populations was similar to their base populations. Nevertheless, the number of rare alleles among the resistant clones had decreased, showing a similar trend at most loci and for most comparisons. Possible implications for breeding strategies are discussed.     

Two conifer species native to Patagonia threatened by Phytophthora austrocedri

Phytophthora austrocedri is a pathogen of Austrocedrus chilensis causing “Mal del Ciprés” (cypress sickness) in Patagonia and killing Juniperus communis in Britain, along with other species in the Cupressaceae. The evident association of the pathogen with members of the Cupressaceae makes it necessary to study the susceptibility of other species related to A. chilensis to prevent the emergence of new diseases. The present study tested the pathogenicity of P. austrocedri to three native and endemic conifers in South America: Fitzroya cupressoides, Pilgerodendron uviferum and Araucaria araucana. The results showed that F. cupressoides and P. uviferum were highly susceptible to P. austrocedri. Since these are endangered tree species, the outcome of this study is crucial for governmental agencies, policymakers and stakeholders.     

Temporal variations in endophytic fungal assemblages of Ginkgo biloba L.

We isolated endophytic fungi from living healthy leaves, petioles, and current-year twigs of Ginkgo biloba L. from April to November 2004 with the objective of identifying the dominant endophytic fungal taxa, and monitoring their occurrence and frequency. A total of 9 fungal taxa were identified to the genus level. Diversity measures inferred from the Shannon–Wiener, Morisita–Horn, and S?rensen indices indicated that leaves and petioles harbored more diverse endophytic fungal assemblages than twigs, and that fungal taxa involved in twigs shared less with those in leaves and petioles. Among the organs, the occurrence pattern of overall endophytic fungi differed significantly, and two taxa, Phomopsis sp. and Phyllosticta sp., were the most frequently isolated and thus regarded as the dominant endophytic fungi. Phomopsis sp. was isolated frequently from twigs (84.8%) but rather few from leaves (16.1%) and petioles (24.3%). Phyllosticta sp. was isolated frequently from leaves (72.9%) and petioles (65.7%) but was never isolated from twigs. Temporal changes in relative frequency of total endophytic fungi tended to differ among sampling dates for all three organs. The occurrence of Phyllosticta sp. in both leaves and petioles was first detected in August and peaked in October. Phomopsis sp. was detected in twigs throughout the growing season. These results suggest that the distribution of the two dominant endophytic fungi was organ-specific and differed within seasons.     

Effects of salicylic acid, 1‐aminocyclopropan‐1‐carboxylic acid and methyl jasmonate on the frequencies of endophytic fungi in Quercus serrata leaves

To examine the effects of salicylic acid (SA), the ethylene precursor 1‐aminocyclopropan‐1‐carboxylic acid (ACC) and methyl jasmonate (MJ), which induce defence responses against pathogen in plants, on foliar endophytic fungi, we performed exogenous treatments of Quercus serrata leaves with these chemicals and subsequently isolated endophytic fungi. SA and ACC significantly reduced the isolation frequency of the most dominant fungus, a Xylaria species as identified by sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA. In contrast, the frequency of the second most frequent fungus increased by SA and ACC. A few other fungi were also affected positively or negatively. Shannon diversity indices decreased by all treatments because of the uneven isolation frequencies of the endophytes. Our results imply that host defence responses induced by the chemical treatments reduced the number of infections of some endophytes while the frequency of some other endophytes increased benefiting from the reduction of competitors.     

Culture‐based methods using low‐temperature incubation revealed cold‐adapted fungal endophytes from semiarid forests

Fungal endophytes are an important part of the plant microbiome and participate in maintaining the stability of ecosystems. Culture‐based methods are frequently considered in studying fungal communities because of their advantages in providing cultures for further experiments and their low cost. In comparison with next‐generation sequencing methods, their ability to assess fungal diversity is questionable. Here, we show how using two different incubation temperatures can improve the yield of fungal endophytes by culture‐based methods. Diversity, organ preference and spatiotemporal variability of endophytic fungal communities living within Quercus brantii L. were investigated using two different incubation temperatures. To test the effects of using low‐temperature incubation on diversity estimations, twigs and leaves of Quercus brantii were incubated under 25°C and 4°C conditions. Isolation rate and Hill's series of diversity were calculated for each sample. Model‐based analysis was used to evaluate the effects of (a) incubation temperature, (b) organ type and (c) spatiotemporal variations on fungal isolation rate, fungal diversity and community composition. Our results showed a strong organ specificity and temporal dynamics among endophytic fungal communities, but the location of the forest stand had only a limited effect on fungal community composition. Low‐temperature incubation enabled us to improve culture‐based methods by discovering the diversity of cold‐adapted fungal endophytes and a significantly different part of the endophytic communities that is usually missed by customary culturing temperature. The isolation of cold‐adapted endophytes from a semiarid environment opens a new window to further studies on this kind of fungi in such ecosystems.     

The penetration rate of laser pulses transmitted from a small-footprint airborne LiDAR: a case study in closed canopy, middle-aged pure sugi (Cryptomeria japonica D. Don) and hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) stands in Japan

Penetration rates of laser pulses transmitted from a small-footprint airborne light detection and ranging (LiDAR) system in closed-canopy, middle-aged (40–50 years old) hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) and sugi (Cryptomeria japonica D. Don) plantations that had similar levels of canopy openness were studied. The number of transmitted pulses was 107 427 points/ha for the hinoki cypress stand and 122 883 points/ha for the sugi stand. The penetration rates of the first (P_f) and second pulses (P_s) that reached the ground were 0.1% and 1.0% in the hinoki cypress stand, and 2.6% and 5.5% in the sugi stand, respectively. Penetration rates for the total number of pulses (P_f+s) that reached the ground within each stand were 1.1% and 8.1%, respectively, and values for P_f+s, P_f, and P_s for sugi and hinoki cypress stands were significantly different (P < 0.001). The results of this study indicated that the generation of accurate digital terrain models in dense hinoki cypress stands with complex topographies using only the data from laser pulses with poor laser penetration rates is likely to be difficult.     

Agrobacterium pusense,a new plant tumour‐inducing pathogen isolated from Lawson cypress

Lawson cypress (Chamaecyparis lawsoniana), an important landscape tree, is widely planted in gardens and parks throughout Iran. Crown gall disease on Lawson cypress trees was observed in Sari and Juybar Counties, Mazandaran province, northern Iran, in 2017. Isolation from galls on potato dextrose agar (PDA) containing CaCO₃ yielded bacterial colonies, the predominant types of which were purified and selected for characterization. The isolates were Gram‐negative, oxidase positive, able to grow in 2% NaCl and produced 3‐ketolactose. They hydrolysed esculin, casein and arbutin but not starch, gelatin or Tween 80. Two representative isolates were selected for PCR amplification and sequencing of DNA gyrase subunit B (gyrB) gene. In the phylogenetic tree based on the partial sequence of the gyrB gene, isolates KH1 and KH2 clustered with Agrobacterium pusense. The pathogenicity of all isolates was confirmed by inoculation on Jimsonweed (Datura stramonium) and carrot discs (Daucus carota). Confirmation of the presence of genes involved in pathogenicity was made by performing PCR with the virD2A/virD2C and VCF/VCR primer pairs which resulted in amplification of the expected 224 and 730 bp fragments in all studied isolates, respectively. A. pusense was therefore identified as the causal agent of crown and stem gall of Lawson cypress. This appears to be the first report on the natural occurrence of crown gall disease on Lawson cypress and the first record of a plant disease caused by A. pusense.     

Performance and quality of <Emphasis Type="Italic">Cupressus sempervirens</Emphasis> L. mini-plug seedlings under reduced photoperiod

Short-day treatment has been used for some conifer species to improve planting stock quality. The aim of this paper was to study the effect of short-day treatment on performance and quality of young seedlings of Italian cypress (Cupressus sempervirens L.), a conifer species abundant in the Mediterranean area. Seeds of Italian cypress were sown in mini-plug cavities. After 5 weeks of growth, seedlings were subjected to four different photoperiods: (a) 14 h (control), (b) 8 h, (c) 6 h, and (d) 4 h for a period of 1–3 weeks, before they were transplanted to bigger size containers. Reducing photoperiod did not result in bud formation or shoot inhibition although it increased the number of lateral shoots. Seedlings subjected to 6-h photoperiod for 2 weeks during pre-cultivation in mini-plugs had improved root characteristics, giving them a higher potential for outplanting success, especially in Mediterranean areas.