First Report of Root Rot Caused by Dactylonectria torresensis on Bletilla striata (Baiji) in Yunnan, China

Bletilla striata (Thunb.) Rchb.f. (Orchidaceae family, known as Baiji in Chinese) is an endangered plant species with important medicinal value in China. Bletilla striata plants with symptoms of wilting, leaf yellowing and rotting on underground parts were found in Shizong (24.82822 N; 103.99084 E), Yunnan Province, China in July 2016. In the following years, this disease occurred and became prevalent when high temperature and high humidity prevailed in the fields from May to August. The incidence of the disease varied from 45 to 75%, with yield losses of 40 to 65% in different B. striata fields. To identify the causal agent of the disease, symptomatic vascular tissue fragments were soaked in 2% sodium hypochlorite for 2 min, rinsed twice with sterile distilled water, and then placed on 4% (w/v) potato dextrose agar (PDA) plates. The plates were incubated at 26°C in 12h light/dark for three days. Mycelia grown from the edges of the plant fragments were transferred to PDA plates and incubated at 26°C in 12h light/dark. After three days, hyphal tips were isolated from the edge of the colonies to PDA plates. Three hyphal-tip isolates from different plants were further studied. The colonies of these three isolates were dark red, with cottony mycelia of moderate density. Hyphae were transparent and branched. Numerous hyphae anastomosed frequently and formed hyphal coils. For further morphological analysis, sporulation was induced as described by Cabral et al. (2012) and Lombard et al. (2014). Macroconidia were abundant, 37.2 to 44.0 µm × 5.2 to 8.7 µm based on the measurement of 20 conidia from each isolate. Ascospores divided into two cells of equal size, ellipsoid to oblong-ellipsoid, 12.5 to 14.8 µm × 4.8 to 5.9 µm based on the measurement of 20 spores from each isolate. Conidiophores simple or complex, sporodochial. Simple conidiophores arising laterally or terminally from aerial mycelium, solitary to loosely aggregated, unbranched or sparsely brached, more or less cylindrical. These morphological characteristics were consistent with the description of Dactylonectria spp. by Cabral et al. (2012) and Lombard et al. (2014). From one isolate, the internal transcribed spacer (ITS) region of ribosomal DNA and the beta-tubulin (tub2) gene were amplified by polymerase chain reaction (PCR) using the primer pairs ITS1/ITS4 (White et al. 1990) and T1/Bt-2b (Cabral et al. 2012), respectively. PCR products were sequenced and deposited in GenBank with accession numbers MH458779 (ITS) and MH626485 (tub2). BLAST search revealed that both sequences showed 99 to 100% homology with the corresponding sequences of previously identified D. torresensis isolates. Specially, MH458779 shares 100% identity with the entire 463-base pair (bp) sequence of KP411806, the ITS sequence of a D. torresensis isolate identified from olive trees (Nigro et al. 2019); MH626485 shares 99% identity with the entire 320-bp sequence of KP411801, the tub2 sequence of the same olive tree isolate. In addition, the entire 609-bp sequence of MH626485 shares 99% identity with JF735478, the tub2 sequence of a D. torresensis isolate identified from grapevines (Cabral et al. 2012). To test the pathogenicity of the fungus, plants of B. striata in plastic pots filled with sterilized nursery soil were inoculated with each of the three isolates by placing a fungal-colonized wheat kernel adjacent to each health plant. Plants inoculated with noncolonized wheat kernels were used as controls. Plants in three pots (replicates), with one plant per pot, were inoculated by each isolate. The pots were maintained in a greenhouse with a 12h photoperiod at 25°C. Ten days after inoculation, black necrotic lesions identical to those observed in the field were evident on the roots of all inoculated plants. Using the same methods described above, fungi with identical morphologies as described above were isolated from lesions caused by each of the three isolates. The control plants remained healthy, and no fungus was re-isolated. This completed Koch’s postulates. Based on the morphological characteristics and molecular identification, the pathogen was determined to be D. torresensis. To our knowledge, this is the first report of D. torresensis causing root rot of B. striata in Yunnan, China. It is important to further study the impacts of this new disease on B. striata production in China.

First Report of Necrotic Leaf Spot Caused by Plectosphaerella cucumerina on Lamb's Lettuce in Southern Italy

During spring 2013, lamb's lettuce plants (Valerianella locusta) cv. Calarasi (Rijk Zwaan) in a commercial greenhouse in Sele Valley (Salerno Province, southern Italy) exhibited small, black-brown, irregular spots (1 mm2) that became necrotic, enlarged, and coalesced. The spots were mostly on the tips of leaves, and were surrounded by a yellow halo. The disease was severe under greenhouse conditions of 60 to 90% RH and maximum air temperature of 26°C, and affected up to 70% of the plants. The greenhouses covered an area of ~3,000 ha where many salad species are grown. Tissue fragments were excised from symptomatic leaves, sterilized by sequential dipping in 70% ethanol for 30 s and in 1% NaOCl for 30 s, rinsed in sterilized distilled water, and placed in 9-cm-diameter petri dishes containing potato dextrose agar (PDA) medium amended with streptomycin sulfate (0.1 g/liter). The plates were incubated at 24°C in the dark. A fungus was isolated consistently from infected leaf tissue after 4 days. Each colony was whitish to orange. Mycelium was hyaline, branched, septate, 3 to 4 μm wide, with numerous anastomosis-forming hyphal coils. Conidiophores were solitary, hyaline, smooth, thin-walled, unbranched or rarely irregularly branched. Conidiogenous cells were phialidic, determinate, discrete, smooth, solitary, and formed on hyphal coils. Phialides were aseptate or occasionally 1-septate near the base. Conidia (n = 100) were ellipsoidal, hyaline, smooth, septate or aseptate, and 6.6 ± 0.9 × 2.8 ± 0.4 μm. On the basis of morphological criteria (3), the fungus was ascribed to Plectosphaerella cucumerina (L.) Laterr. (anamorph Plectosporium tabacinum). An aliquot (50 ng) of genomic DNA extracted from mycelium of five cultures obtained by monosporic isolation on PDA was used as template for a PCR reaction with primers ITS5/ITS4, specific for the ITS 5.8S rDNA region of fungi (3). The 500-bp sequences amplified from the five isolates were identical, and the sequence of isolate Val-2 was submitted to GenBank (KF753234). Sequence analysis with BLASTn showed 100% identity of this sequence to the ITS-5.8S rDNA sequences of 11 isolates of P. cucumerina in GenBank. Three isolates were selected for pathogenicity tests on the lamb's lettuce cv. Calarasi. Before planting, seeds were surface-disinfected in 1% NaOCl and rinsed with sterilized distilled water. Plants (35 days old, 30 plants tested/isolate) were grown in 0.7-liter pots filled with a sterilized (autoclaved at 112°C for 1 h on each of two consecutive days) mixture of soil:sand:perlite (2:1:1), and inoculated by spraying the leaves with a spore suspension (106 CFU/ml, ~3 ml applied/plant) of each isolate prepared from 7-day-old cultures on PDA. As a control, five plants were sprayed with sterilized water. All plants were incubated in a growth chamber at 90% RH with a 12-h photoperiod at 24°C. Leaf spots typical of those on the original symptomatic plants appeared 7 to 10 days after inoculation on all inoculated plants. No symptoms were observed on control plants. P. cucumerina was re-isolated only from symptomatic leaves, as described for the original isolations. P. cucumerina has been associated with root and collar roots of some horticultural crops (1), and a leaf spot on Diplotaxis tenuifolia (2), often grown in rotation with lamb's lettuce in southern Italy. To our knowledge, this is the first report of P. cucumerina as a pathogen of V. locusta in Italy or elsewhere. The disease caused economic loss to lamb's lettuce, primarily used in Italy in fresh-cut, mixed salads. References: (1) A. Carlucci et al. Persoonia 28:34, 2012. (2) A. Garibaldi et al. Plant Dis. 96:1825, 2012. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Observations on the mycorrhizal status of Polygonum viviparum in the Polish Tatra Mts. (Western Carpathians)

<em>Polygonum viviparum</em> is one of very few herbaceous plants known to form ectomycorrhiza; in the Tatra Mts. it is one of dominants in the alpine zone, but also descends down to the feet of the massif. Specimens of this plant were collected from 5 sites at the altitude range 900– 2150 m, from granite and limestone. It allowed an estimation of the ectomycorrhizal diversity as well as preliminary ecological observations. Roots were also stained in order to check potential presence of arbuscular mycorrhizal colonization. Ectomycorrhizae were present in all specimens (with 2–5 morphotypes observed on single plants). In total, 17 morphotypes were observed and briefly described. The most widespread were the mycorrhiza of <em>Cenococcum geophilum<em> and a brightly coloured morphotype resembling the ectomycorrhizae of <em>Russula</em> sp. No important differences in ectomycorrhizal colonization between low and high localities were found. Observed general differences in abundance and diversity of mycorrhiza in <em>P. viviparum</em> between sites could most probably be connected with plant community composition (presence/absence of ectomycorrhizal shrubs maintaining ectomycorrhizal fungi), although mycorrhizae were present also in sites devoid of other ectomycorrhizal plants. Structures associated to arbuscular colonization (vesicles, hyphal coils) were occassionally observed, but without formation of arbuscules.

Fungal associations in gametophytes and young sporophytic roots of the fern Nephrolepis exaltata

Fungal associations in gametophytes and young sporophytic roots of the fernNephrolepis exaltataInformation is limited on the presence of endophytic fungal associations in green gametophytes and young sporophytes of extant ferns. Nothing is known about their presence in Polypodiales, the largest order among extant ferns. We screened chlorophyllous gametophytes and young sporophytes ofNephrolepis exaltata(L.) Schott., (Lomariopsidaceae, Polypodiales) growing naturally on soil, brick and coir for the presence of fungal endophytes. Gametophytes and young sporophytes growing on different substrates were invariably colonized by septate endophytic fungi. Hyaline or brown, regularly septate, inter- or intracellular hyphae with moniliform cells or microsclerotia characterized septate endophytic fungi. However, only the roots of young sporophytes growing on soil and bricks harboured arbuscular mycorrhizal (AM) fungi. The AM morphology conformed to the intermediate type with intracellular hyphal coils, arbusculate coils and intercellular hyphae. No AM fungal spores could be retrieved from the soil on which gametophytes and young sporophytes were growing. The observations in this study support the idea that the septate fungal endophytes could confer an ecological advantage on colonized individuals, especially on nutrient deficient substrates.

Cortical colonisation is not an absolute requirement for phosphorus transfer to plants in arbuscular mycorrhizas formed by Scutellospora calospora in a tomato mutant: evidence from physiology and gene expression

Arbuscules in Arum-type arbuscular mycorrhizas (AM), formed intracellularly in root cortical cells, are generally believed to be the most important and defining characteristics of the symbiosis as sites for phosphorus (P) and carbon (C) exchange. We used a Pen + Coi– phenotype (penetration of epidermal and exodermal root cells but not arbuscule formation) formed in rmc (reduced mycorrhizal colonisation) mutant tomato (Lycopersicon esculentum Mill.) by Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders to determine whether the fungus is capable of transferring P from soil to plant and whether there is concurrent upregulation of AM-inducible orthophosphate (Pi) transporter gene expression in the roots. Our physiological data showed that colonisation of outer root cell layers is sufficient for P transfer from S. calospora to tomato. This transfer of P was supported by increased expression of the Pi transporter genes, LePT3 and LePT5, known to be upregulated in AM interactions. We conclude that cortical colonisation and formation of arbuscules or arbusculate hyphal coils is not an absolute prerequisite for P transfer in this symbiosis.

Atractiellomycetes belonging to the ‘rust’ lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids

Distinctive groups of fungi are involved in the diverse mycorrhizal associations of land plants. All previously known mycorrhiza-forming Basidiomycota associated with trees, ericads, liverworts or orchids are hosted in Agaricomycetes, Agaricomycotina. Here we demonstrate for the first time that Atractiellomycetes, members of the ‘rust’ lineage (Pucciniomycotina), are mycobionts of orchids. The mycobionts of 103 terrestrial and epiphytic orchid individuals, sampled in the tropical mountain rainforest of Southern Ecuador, were identified by sequencing the whole ITS1-5.8S-ITS2 region and part of 28S rDNA. Mycorrhizae of 13 orchid individuals were investigated by transmission electron microscopy. Simple septal pores and symplechosomes in the hyphal coils of mycorrhizae from four orchid individuals indicated members of Atractiellomycetes. Molecular phylogeny of sequences from mycobionts of 32 orchid individuals out of 103 samples confirmed Atractiellomycetes and the placement in Pucciniomycotina, previously known to comprise only parasitic and saprophytic fungi. Thus, our finding reveals these fungi, frequently associated to neotropical orchids, as the most basal living basidiomycetes involved in mycorrhizal associations of land plants.

The mycoheterotroph Arachnitis uniflora has a unique association with arbuscular mycorrhizal fungi

Achlorophyllous plants that are dependent on an association with fungi linked to photosynthetic plants for their carbon source are known as mycoheterotrophs. Arachnitis uniflora Phil., a monotypic member of the monocotyledonous family Corsiaceae, fits this category, as it relies on a glomalean fungus belonging to Glomus Group A for carbon acquisition. Although basic structural features of root colonization have been reported for A. uniflora, the nutrient exchange interface has not been studied. This is the first study to use confocal microscopy, transmission electron microscopy, and cytochemical procedures to study the interface between a glomalean fungus and the roots of a mycoheterotrophic species. Results showed that arbuscules are never formed, and that the “vesicles in bundles” reported earlier are unlike typical glomalean vesicles, in that they form in clusters by the enlargement of hyphal branches and have a complex multilayered wall. The thick inner wall layer consists primarily of β-1,3-glucans (callose) and is surrounded by a thin outer layer of chitin. Each structure is surrounded by host cell wall material and a perifungal membrane, suggesting an involvement in nutrient exchange. The cytoplasm contains a complex of small β-1,3-glucan-containing vacuoles, lipid bodies, endobacteria, and many nuclei. These structures enlarge to occupy most of the cortical cell volume and then degrade, releasing lipids and other materials into the host cell. We suggest that these structures should not be equated with typical glomalean vesicles but are unique structures that may be involved, along with the hyphal coils, in nutrient acquisition by the host.

Basidiomycetous endophytes in New Zealand Aneuraceae (simple thalloid liverworts, Metzgeriidae) and the derived status of the monotypic genus Verdoornia

The ultrastructure of endophytic basidiomycetes in two New Zealand species of Aneura and the endemic genus Verdoornia is compared with that in northern hemisphere members of the Aneuraceae. The discovery of a basidiomycete colonization in Verdoornia and a reappraisal of earlier morphological data confirms molecular evidence that this is a derived genus nested within the Aneuraceae rather than a primitive isolated taxon, as considered previously. Throughout the Aneuraceae, the basidiomycetes exhibit similar colonization patterns; initial proliferation of dikaryotic, non-clamp-forming hyphal coils within the host cells is followed by senescence and aggregation of collapsed hyphae into large masses. Multiple cycles of colonization are common. Dolipores with imperforate parenthesomes indicate that the fungi are all heterobasidiomycetes. In line with earlier cross-colonization experiments showing complete host specificity, differences in hyphal diameters and dolipore dimensions suggest that the fungi belong to different taxa. The New Zealand aneuracean fungi have multilayered walls similar to their northern hemisphere counterparts. Within the Aneuraceae there appears to be a trend from very limited or no endophyte colonization in Riccardia to colonization of the entire thallus in Cryptothallus. The cytology of the fungal associations in Aneura and Verdoornia is highly suggestive of mixotrophy.