Ampelomyces strains isolated from diverse powdery mildew hosts in Japan: Their phylogeny and mycoparasitic activity, including timing and quantifying mycoparasitism of Pseudoidium neolycopersici on tomato

A total of 26 Ampelomyces strains were isolated from mycelia of six different powdery mildew species that naturally infected their host plants in Japan. These were characterized based on morphological characteristics and sequences of ribosomal DNA internal transcribed spacer (rDNA-ITS) regions and actin gene (ACT) fragments. Collected strains represented six different genotypes and were accommodated in three different clades of the genus Ampelomyces. Morphology of the strains agreed with that of other Ampelomyces strains, but none of the examined characters were associated with any groups identified in the genetic analysis. Five powdery mildew species were inoculated with eight selected Ampelomyces strains to study their mycoparasitic activity. In the inoculation experiments, all Ampelomyces strains successfully infected all tested powdery mildew species, and showed no significant differences in their mycoparasitic activity as determined by the number of Ampelomyces pycnidia developed in powdery mildew colonies. The mycoparasitic interaction between the eight selected Ampelomyces strains and the tomato powdery mildew fungus (Pseudoidium neolycopersici strain KTP-03) was studied experimentally in the laboratory using digital microscopic technologies. It was documented that the spores of the mycoparasites germinated on tomato leaves and their hyphae penetrated the hyphae of Ps. neolycopersici. Ampelomyces hyphae continued their growth internally, which initiated the atrophy of the powdery mildew conidiophores 5 days post inoculation (dpi); caused atrophy 6 dpi; and complete collapse of the parasitized conidiphores 7 dpi. Ampelomyces strains produced new intracellular pycnidia in Ps. neolycopersici conidiophores ca. 8–10 dpi, when Ps. neolycopersici hyphae were successfully destroyed by the mycoparasitic strain. Mature pycnidia released spores ca. 10–14 dpi, which became the sources of subsequent infections of the intact powdery mildew hyphae. Mature pycnidia contained each ca. 200 to 1,500 spores depending on the mycohost species and Ampelomyces strain. This is the first detailed analysis of Ampelomyces strains isolated in Japan, and the first timing and quantification of mycoparasitism of Ps. neolycopersici on tomato by phylogenetically diverse Ampelomyces strains using digital microscopic technologies. The developed model system is useful for future biocontrol and ecological studies on Ampelomyces mycoparasites.

Variation in UV-mediated damage recovery among Pseudoidium neolycopersici isolates: possible mechanisms.

The cryptochrome/photolyase family (CPF) consists of a diversified class of flavoproteins that are evolutionarily related. Although their domain architecture is highly conserved, they perform entirely different physiological functions. Previous studies have confirmed the presence of a functional photolyase in Pseudoidium neolycopersici, which repairs UV-C-induced DNA damage by using near UV-A/blue light as an energy source. Similar doses of UV-C treatment followed by incubation with dark or blue light was tested on conidia germination of five isolates of P. neolycopersici collected from different regions of Norway and the Netherlands. The results showed variations in the effect of UV on germination and germination recovery under blue light incubation after UV treatment. Evolutionary studies confirmed that P. neolycopersici photolyase is highly conserved among different isolates of P. neolycopersici and among different species. All CPF members have a core domain consisting of an identical cofactor Flavin adenine dinucleotide (FAD), and an additional photoantenna chromophore. An amino acid sequence analysis demonstrated that CPF members have highly conserved C-terminals compared with their N-terminals, as FAD binds in the C-terminal region. When compared with other CPF members, varying lengths of N- and C-terminals were noticed in P. neolycopersici photolyase and putative cryptochrome, respectively. Further research using comparative genomics targeting pyrimidine base composition, the role of regulatory elements including promotor architecture, and the characterization of optical properties of native photolyase among isolates may help to explain the differences in the biological responses of conidia germination of P. neolycopersici treated with UV.

First Report of Powdery Mildew (Pseudoidium neolycopersici) on Croton (Codiaeum variegatum var. pictum) in China

Croton (Codiaeum variegatum (Linn.) var. pictum (Lodd.)) is an ornamental plant commonly grown in southern China. In March 2014, severe powdery mildew infections were observed on crotons in gardens of Hainan University (20.1°N and 110.3°E), Haikou, Hainan province. Disease incidence was estimated in a random batch of 100 plants in three replicates, with the average value approaching 80%. Symptoms first appeared as white circular patches on the adaxial surface and expanded to the abaxial surface, petioles, and stems. The top leaves were the most affected. Upper surfaces of the infected leaves were covered by white, dense mycelia. As the disease progressed, infected leaves turned purple on the lower surfaces and curly before becoming necrotic and abscising from the plant. Powdery mildew was more severe in shaded environments, especially during rainy or foggy weather in early spring. Two hundred conidiophores and conidia were observed microscopically. The conidiophores were straight or occasionally flexuous, 62.3 to 127.6 × 6.2 to 10.2 μm, consisting of two to three straight cells. Conidia were born in solitary on the top of conidiophores. Conidia were hyaline, ellipsoidal, 26.4 to 42.2 × 11.7 to 23.4 μm (average 32.5 × 16.5 μm), contained no distinct fibrosin bodies, and produced a subterminal germ tube. The wrinkling pattern of the outer walls of older conidia was angular or reticulated. Appressoria were single and multilobed. Cleistothecia were not observed. Based on morphological characteristics, the fungus was identified as Oidium neolycopersici (2), which was recently renamed Pseudoidium neolycopersici (L. Kiss) (3). The identity was confirmed by sequence analysis. Genomic DNA was extracted from the foliar powdery mildew colonies using Chelex-100 (Bio-Rad, Shanghai, China). The rDNA internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 (5). The ITS sequence of the representative isolates C01 (GenBank Accession No. KJ890378.1) and four other powdery mildew samples collected from crotons in Hainan University was 100% identical to that of P. neolycopersici isolates from tomato plants such as JQ972700 and AB163927. Inoculations were made by gently pressing diseased leaves onto leaves of five healthy plants of croton and tomato (‘Money maker’). Five non-inoculated croton and tomato plants served as controls. Inoculated and non-inoculated plants were maintained in an incubator at 25°C with a 12-h photoperiod. After eight days, typical powdery mildew symptoms developed on 93% of the inoculated plants, while no symptom developed on the non-inoculated plants. The pathogenicity tests were repeated three times. The same fungus was always re-isolated from the diseased tissue according to Koch's postulates. The pathogenicity tests further confirmed that the pathogen from crotons is P. neolycopersici (Basionym. Oidium neolycopersici (KJ890378.1)), which is commonly known as the tomato powdery mildew. P. neolycopersici is also a pathogen of Normania triphylla (1) and papaya (4). To our knowledge, this is the first report of P. neolycopersici infecting croton. The avenue of this pathogen entering gardens of Hainan University remains unknown. The gardens are located far away from tomato farms. Also no symptom of powdery mildew on croton was observed during surveys in other locations in Haikou. The origin of the pathogen warrants additional research. References: (1) D. Delmail et al. Mycotaxon 113:269, 2010. (2) L. Kiss et al. Mycol. Res. 105:684, 2001. (3) L. Kiss et al. Mycol. Res. 115:612, 2011. (4) J. G. Tsay et al. Plant Dis. 95:1188, 2011. (5) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.