Microscopic and ultramicroscopic anatomical characteristics of root nodules in Podocarpus macrophyllus during development

To understand the morphological and structural characteristics of root nodules in Podocarpus macrophyllus and their development, this study prepared P. macrophyllus root nodule samples at the young, mature, and senescent stages. Optical microscopy and transmission electron microscopy (SEM) revealed that new nodules can be formed on roots and senescent nodules; new nodules formed on the roots are nearly spherical and have an internal structure similar to finite nodules; new nodules on senescent nodules are formed by extension and differentiation of the vascular cylinder of the original nodules; and these new nodules are nested at the base of the original nodules, which create growth space for new nodules by dissociating the cortical tissue; clusters of nodules are formed after extensive accumulation, and the growth pattern is similar to that of infinite nodules; the symbiotic bacteria of P. macrophyllus root nodules mainly invade from the epidermal intercellular space of the roots and migrate along the intercellular space of the nodule cortex; infected nodule cortex cells have a well-developed inner membrane system and enlarged and loose nuclei; and unique Frankia vesicles, and rhizobia cysts, and bacteriophages can all develop. Compared with common leguminous and nonleguminous plant nodules, P. macrophyllus root nodules are more complex in morphology, structure and composition. From the perspective of plant system evolution, the rhizobium nodules in leguminous angiosperms and Frankia nodules in nonleguminous angiosperms are most likely two branches derived from the nodules in gymnosperms, such as P. macrophyllus. The conclusions of this study can provide a theoretical basis for the developmental biology of P. macrophyllus root nodules and the evolutionary pattern of plant symbionts.

First report of Fusarium concentricum causing wilt on Podocarpus macrophyllus in China

Podocarpus macrophyllus (Thunb.) D. Don is used in many fields, including landscape, medicine, and forest interplanting. In July 2019, shoot blight was observed on P. macrophyllus at three nurseries in Harbin, China. Approximately 15% of plants had symptoms of the disease, which included rapid, synchronized death of leaves on individual branches. Eventually the whole plant wilted. Leaves and stems turned dark blue to brown. Ten infected vascular tissue samples from 10 individual plants were surface-disinfested in 0.5% NaOCl for 5 min, rinsed 3 times in sterile distilled water, and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 26°C. Six similar fungal isolates from ten samples were isolated and subcultured. Single-conidium isolates were generated with methods reported previously (Leslie and Summerell 2006). Colonies on PDA consisted of densely floccose aerial hyphae with light yellow and pinkish pigments. Microconidia were oval to obovoid or allantoid, 3.8 to 11.8 μm in length and 2.8 to 4.6 μm in width, mostly non-septate on carnation leaf agar (CLA). Macroconidia were naviculate-to-fusiform slender, 24.9 to 57.2 μm in length and 2.8 to 4.5 μm in width with 3- to 5- septate, with a beaked apical cell and a foot-shaped basal cell. According to these morphological characteristics, all isolates were identified as Fusarium spp. (Aoki et al. 2001 ). Genomic DNA was extracted from a representative isolate LHS1. The internal transcribed spacer regions (ITS), translation elongation factor 1-alpha gene (TEF-1ɑ) and β-tubulin (TUB2) gene were amplified using the primers ITS1 and ITS4 (Yin et al. 2012)，EF1-728F/EF1-986R (Carbone and Kohn 1999) and T1/Bt2b (Glass and Donaldson 1995), respectively. DNA sequences of LHS1 were deposited in GenBank (accession nos. MT914496 for ITS, MT920920 for TEF-1ɑ and MT920921 for TUB2, respectively). MegaBLAST analysis of the ITS, TEF-1a, and TUB2 sequences indicated 100%, 97.7% and 100% similarity with Fusarium concentricum isolate CBS 450.97 (accession no. MH862659.1 for ITS, MT010992.1 for TEF-1a, and MT011040.1 for TUB2, respectively). To determine pathogenicity, P. macrophyllus plants were grown in 10-cm pots containing a commercial potting mix (five plants/pot). At the 10 to 12 leaf stage, 10 healthy plants (2 pots) were inoculated by spraying 5 ml of a conidial suspension (4×106 spores/ml) onto every plant. Ten plants treated with sterile distilled water served as a control. The test was repeated twice. All plants were placed in a humidity chamber (>95% RH, 26℃) for 48 h after inoculation and then transferred to a greenhouse at 22/28°C (night/day). All inoculated wilted with leaves and stems turning dark blue to brown 15 days after inoculation. No symptoms were observed on the control plants. The fungus was re-isolated and confirmed to be F. concentricum according to morphological characteristics and molecular identification. To our knowledge, this is the first report of F. concentricum on P. macrophyllus in world. The disease caused a large number of plants to wilt and die, seriously impacting the ability of the horticulture industry to produce P. macrophyllus. Although this pathogen causes leaf and shoot blight symptoms, it is not clear if the pathogen is also a vascular wilt disease. The occurrence of the new disease caused by F. concentricum highlights the importance of developing management strategies to protect P. macrophyllus.

Vegetative propagation of an ornamental conifer: some techniques in podocarpo cutting

ABSTRACT: The asexual propagation of conifers, such as Podocarpus macrophyllus (Thunb.) Sweet, faces difficulties. Among the factors that can improve the efficiency of asexual propagation are the types of cuttings used and lesions performed at the base of the cuttings, and the use of plant regulators, such as indolebutyric acid (IBA). This study determined the types of lesions to be performed at the cutting base, the type of cuttings used, the method to convey IBA at the cutting base, and the concentration of IBA required to obtain a proper rooting condition for the species. Two experiments were performed from March to July, 2018, in randomized blocks and with a factorial scheme, as follows: 2 x 4 (IBA application in solution and talc x IBA at concentrations of 0, 500, 1,000, and 1,500 mg L-1 IBA); and 2 x 3 (basal and apical cuttings x bevel, wedge, and horizontal lesions at the cutting base). In both experiments, the cuttings used were arranged on a masonry bed containing medium-textured sand. After 120 days of experimentation, the study concluded that the rooting percentage was favored by the use of basal cuttings with wedge lesions at the cutting base and treated with IBA in solution at a concentration of approximately 550 mg L-1.

A new cystoid nematode, Cryphodera japonicum n. sp. (Nematoda: Heteroderidae), from the rhizosphere of Japanese Podocarpus macrophyllus

Summary Cryphodera japonicum n. sp., detected at Ningbo port, China, from the rhizosphere of imported Japanese Podocarpus macrophyllus, is described. The new species is characterised by females with a globose body, protruding vulval lips, slightly concave anus-vulva profile, a stylet length of 37.2 (31.1-41.3) μm and a vulva-anus distance of 38.1 (34.7-44.1) μm. Males possess two lip annuli, a stylet length of 27 (22.5-30.6) μm long, three lines in lateral fields and a spicule 21.5 (19.1-23.0) μm long. Second-stage juveniles have bodies 543 (506-588) μm long, three lip annuli, a stylet 31.7 (29.2-34.5) μm long, three lines in the lateral fields, a conoid tail with a narrow rounded terminus, and a relatively long hyaline region occupying half of the tail length. Phylogenetic analyses based on the D2-D3 expansion segments of the 28S, ITS, partial 18S rRNA, and COI gene revealed the unique position of this species with other heteroderid nematodes, supporting its status as a new species of Cryphodera. The new species showed a close relationship with C. brinkmani.

Phyllocnistis podocarpa sp. nov. (Lepidoptera, Gracillariidae), a buddhist pine leaf-miner from Japan: taxonomy, DNA barcodes, damage and parasitoids

Phyllocnistis podocarpa sp. nov., is described from mines in Podocarpus macrophyllus (Family Podocarpaceae). The host plant P. macrophyllus, also known as buddhist pine on the IUCN Red List, is a noticeable garden plant and thus of high economic value. Buddhist pine has been introduced to many other countries from its native habitat in southern Japan. Special attention has been paid for it during the overseas import in China. The morphology of the pupae of P. podocarpa, particularly the frontal process of the head and the spine clusters on terga, ones of the most useful diagnostic characters for species identification of Phyllocnistis on morphology, is demonstrated using SEM. Two parasitoid species of Eulophidae (Hymenoptera) are identified and illustrated. COI barcode sequences are provided along with a Neighbor Joining Tree covering related species for aiding identification.