Succession patterns of fungi associated to wound-induced agarwood in wild Aquilaria malaccensis revealed from quantitative PCR assay

Aquilaria malaccensis produces agarwood in response to wounding and fungal attack. However, information is limited regarding Aquilaria‘s interaction with its diverse fungal community. In this study, time-related changes of three natural fungal colonizers in two wounded wild A. malaccensis were tracked, beginning a few hours after wounding up to 12 months. Using species-specific primers derived from their nrITS sequences in quantitative real-time PCR (qPCR), we quantified the amount of Cunninghamella bainieri, Fusarium solani and Lasiodiplodia theobromae. Because time is a major factor affecting agarwood quantity and quality, 14 wood samples were collected at different time points, i.e., 0–18 h, 2–13 days, 2–18 weeks, and 6–12 months after wounding. qPCR data revealed that the abundance of the three species decreased over time. The fungi were detected in high numbers during the first few hours and days after wounding (40- to 25,000-fold higher levels compared with initial counts) and in low numbers (<1- to 3,200-fold higher than initially) many months later. Consistent with its role in defense response, the accumulation of secondary metabolites at the wounding site could have caused the decline in fungal abundance. Succession patterns of the two trees were not identical, indicating that fungal populations may have been affected by tree environment and wound microclimate. Our results are important for understanding the diversity of microbial community in wild Aquilaria species and their association to wound-induced agarwood formation. Fungi could be secondary triggers to agarwood production in situations where trees are wounded in attempt to induce agarwood.     

Biochemical analysis of reactive oxygen species production and antioxidative responses in unripe avocado (Persea americana Mill var Hass) fruits in response to wounding

We analyzed the production of reactive oxygen species (ROS) and of detoxifying enzymes and enzymes of the ascorbate (ASC) acid cycle in avocado fruit (Pesea Americana Mill cv Hass) in response to wounding. The levels of superoxide anion (O₂ ^?), hydroxyl radicals (OH^.) and hydrogen peroxide (H₂O₂) increased at 15 min and 2 and 15 h post-wounding. Peroxidase (POD) activity had increased to high levels 24 h after wounding; in contrast, catalase and superoxide dismutase (SOD) levels hat decreased significantly at 24 h post-treatment. Basic POD was the major POD form induced, and the levels of at least three apoplastic POD isozymes –increased following wounding. Using specific inhibitors, we characterized one MnSOD and two CuZnSOD isozymes. CuZnSOD activities decreased notably 12 h after treatment. The activities of dehydroascorbate reductase and glutathione reductase increased dramatically following the wounding treatment, possibly as a means to compensate for the redox changes due to ROS production.     

蒙古冰草AmWRKY1-like基因克隆及表达分析

基于转录组测序数据,该研究采用PCR扩增方法从蒙古冰草中克隆AmWRKY1-like的全长cDNA,并对其进行了生物信息学和亚细胞定位分析,为深入研究AmWRKY1-like在蒙古冰草干旱胁迫响应中的调控作用提供理论基础。结果发现:成功克隆得到了AmWRKY1-like基因,其开放阅读边框(ORF)为1 182 bp、编码393个氨基酸,含有典型的WRKY转录因子保守结构域;qRT-PCR表明,在自然干旱和15%PEG-6000模拟干旱下,蒙古冰草叶片中AmWRKY1-like均可诱导表达,较对照呈下调表达趋势;亚细胞定位表明,AmWRKY1-like基因在细胞核中特异表达。     

Distribution of <Emphasis Type="Italic">Petrosavia sakuraii</Emphasis> (Petrosaviaceae), a rare mycoheterotrophic plant,may be determined by the abundance of its mycobionts

Petrosavia sakuraii (Petrosaviaceae) is a rare, mycoheterotrophic plant species that has a specific symbiotic interaction with a narrow clade of arbuscular mycorrhizal (AM) fungi. In the present study, we tested the hypothesis that the distribution and abundance of mycobionts in two P. sakuraii habitats, Nagiso and Sengenyama (central Honshu, Japan), determine the distribution pattern of this rare plant. Nagiso is a thriving habitat with hundreds of P. sakuraii individuals per 100 m², whereas Sengenyama is a sparsely populated habitat with fewer than 10 individuals per 100 m². AM fungal communities associated with tree roots were compared at 20-cm distances from P. sakuraii shoots between the two habitats by molecular identification of AM fungal partial sequences of the small subunit ribosomal RNA gene. The percentage of AM fungal sequences showing over 99 % identity with those of the dominant P. sakuraii mycobionts was high (54.9 %) in Nagiso, but low (13.2 %) in Sengenyama. Accordingly, the abundance of P. sakuraii seems to reflect the proportion of potential mycobionts. It is likely that P. sakuraii mycobionts are not rare in Japanese warm temperate forests since 11.2 % of AM fungal sequences previously obtained from a deciduous broad-leaved forest devoid of P. sakuraii in Mizuho, central Honshu, Japan, were >99 % identical to those of the dominant P. sakuraii mycobionts. Thus, results suggest that the abundant mycobionts may be required for sufficient propagation of P. sakuraii, and this quantitative trait of AM fungal communities required for P. sakuraii may explain the rarity of this plant.     

Comparison of the diversity,composition, and host recurrence of xylariaceous endophytes in subtropical,cool temperate,and subboreal regions in Japan

The diversity, composition, and host recurrence of endophytic fungi in the Xylariaceae were compared in subtropical (ST), cool temperate (CT), and subboreal forests (SB) in Japan based on the 28S ribosomal DNA sequences from fungal isolates. A total of 610 isolates were obtained from the leaves of 167 tree species in three sites, which were classified into 42 operational taxonomic units (OTUs) at the 99 % similarity level of the 28S rDNA sequence. ST, CT, and SB yielded 31, 13, and three OTUs, respectively. The OTU richness, diversity, and evenness of fungal communities were in the order: ST > CT > SB. The 42 OTUs were assigned to nine genera in the Xylariaceae: Xylaria, Annulohypoxylon, Anthostomella, Biscogniauxia, Nemania, Hypoxylon, Muscodor, Daldinia, and Rosellinia. Xylarioid isolates in the subfamily Xylarioideae outnumbered Hypoxyloid isolates in the subfamily Hypoxyloideae in ST and CT, whereas the opposite was found in SB. Sørensen’s quotient of similarity was generally low between the three sites. Host recurrence of fungal OTUs was evaluated with the degree of specialization of interaction network between xylariaceous endophytes and plant species and compared between the three sites. We found that the networks in the three sites showed a significantly higher degree of specialization than simulated networks, where partners were associated randomly. Permutational multivariate analyses of variance indicated that plant family and leaf trait significantly affected the OTU composition in ST, which can account for the specialization of interaction network and host recurrence of xylariaceous endophytes.     

The C-terminal region of OsWRKY30 is sufficient to confer enhanced resistance to pathogen and activate the expression of defense-related genes

WRKY proteins are known to play major roles in defense signaling. We identified a pathogen-inducible and SA-inducible OsWRKY30. Our cDNA clone encodes the C-terminal region (CTR) of OsWRKY30. CTR-OsWRKY30 includes the C-terminal WRKY domain and nuclear localization sequence. CTR-OsWRKY30 was sufficient to bind W-box sequences (TTGACC/T). Over-expression of the CTR-OsWRKY30 resulted in enhanced resistance to pathogens in Arabidopsis and rice. Defense-related genes were constitutively expressed in transgenic Arabidopsis and rice over-expressing CTR-OsWRKY30. Based on promoter transient assays, CTR-OsWRKY30 is sufficient to activate OsPR10a promoter as much as full length OsWRKY30. Taken together, CTR-OsWRKY30 positively regulates defense signaling, thereby resulting in enhanced resistance to pathogens.     

Immune defenses of <Emphasis Type="Italic">Agriotes lineatus</Emphasis> larvae against entomopathogenic nematodes

The present work describes the immune response of wireworm larvae, Agriotes lineatus (L.) (Coleoptera: Elateridae) when challenged with two species of entomopathogenic nematodes, Steinernema feltiae (Filipjev) (Strongyloidea: Steinernematidae) and Heterorhabditis bacteriophora (Poinar) (Rhabiditoidea: Heterorhabditidae). Two main immunological processes including cellular and humoral reactions have been addressed. Total haemocyte counts after infection with H. bacteriophora increased quickly in initial times, but decreased over time post-injection (at 12 and 16 h). Instead, haemocyte numbers after infection with S. feltiae was unchanged in the early stage, but significantly decreased until 16 h post-injection. Plasmatocytes and granulocytes showed more significant changes compared to other haemocytes. The encapsulation response to parasites was significantly different against two nematode species. Particularly, S. feltiae was almost unrecognized by host haemocytes (5.85 % of encapsulated parasites). Assays with H. bacteriophora showed 23.5 % of encapsulated nematodes. From 8 to 12 h after H. bacteriophora infection, an increase in phenoloxidase activity was detected, while in the larvae injected with S. feltiae the enzymatic activity decreased gradually reaching the lowest level 16 h post-injection. This is the first report on the modulation of immune response of wireworm larvae after infection with entomopathogenic nematodes.     

High diversity of root-associated fungi isolated from three epiphytic orchids in southern Ecuador

Knowledge of fungal root-associates is essential for effective conservation of tropical epiphytic orchids. We investigated the diversity of root-associated fungi of Cyrtochilum myanthum, Scaphyglottis punctulata and Stelis superbiens from a tropical mountain rainforest in southern Ecuador, using a culture dependent approach. We identified 115 fungal isolates, corresponding to 49 fungal OTUs, based on sequences of the nrDNA ITS and partial 28S region. Members of Ascomycota were unambiguously dominant (37 OTUs), including Trichoderma sp. as the most frequent taxon. Members of Basidiomycota (Agaricales and Polyporales) and Mucoromycota (Umbelopsidales and Mortierellales) were also identified. Four potential mycorrhizal OTUs of Tulasnellaceae and Ceratobasidiaceae were isolated from C. myanthum and S. superbiens. Fungal community composition was examined using Sørensen and Jaccard indices of similarity. Alfa diversity was significantly different between C. myanthum and S. superbiens. No difference in beta diversity of the fungal communities between the 3 orchid species and the collecting sites was detected. The study revealed a high diversity of fungi associated with orchid roots. Our results contribute to a better understanding of specific relationships between epiphytic orchids and their root-associated fungi.     

The return of an experimentally N-saturated boreal forest to an N-limited state: observations on the soil microbial community structure,biotic N retention capacity and gross N mineralisation

Background and aims

To find out how N-saturated forests can return to an N-limited state, we examined the recovery of biotic N sinks under decreasing N supply.

Methods

. We studied a 40-year-old experiment in Pinus sylvestris forest, with control plots, N0, three N treatments, N1-N3, of which N3 was stopped after 20 years, allowing observation of recovery.

Results

In N3, the N concentration in foliage was still slightly elevated, but the N uptake capacity of ectomycorrhizal (ECM) roots in N3 was no longer lower than in N0. Per area the amount of a biomarker for fungi, here mainly attributed ECM, was higher in N3 and N0 than in N1 and N2. Retention of labeled ¹⁵NH₄ ⁺ by the soil was greater in the control (99 %) and N3 (86 %), than in N1 (45 %) and N2 (29 %); we ascribe these differences to biotic retention because cation exchange capacity did not vary. Gross N mineralisation and retention of N correlated, negatively and positively, respectively, with abundance of ECM fungal biomarker.

Conclusions

. The results suggest a key role for ECM fungi in regulating the N cycle. We propose, in accordance with plant C allocation theory, that recovery is driven by increased tree below-ground C allocation to ECM roots and fungi.