Tingyu Duan

Microdochium tabacinum, Confirmed as a Pathogen of Alfalfa in Gansu Province, China

A crown and root rot complex was detected in the alfalfa (Medicago sativa Longdong) fields of Huanxian County. The symptoms of the diseased plants were characterized, and 11 fungal species were obtained from the roots. These fungi included isolates that resembled the genus Microdochium. An isolate of this type, designated MP313, was proven to infect alfalfa, fulfilling Kochs postulates. Isolate MP313 was examined by microscopy and the morphological characteristics indicated that it was similar to members of the genus Microdochium. Sequence analyses of the 28S large subunit as well as the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) of MP313 revealed 98 to 100% similarity to the corresponding regions of M. tabacinum. A polymerase chain reaction assay based on the ITS region of the rDNA was developed to amplify a 304-bp fragment from DNA concentrations as low as 20 fg/μl, which was sensitive enough to detect isolate MP313 in diseased root samples. Taken together, these results confirmed that M. tabacinum was one of a complex of fungi associated with crown and root rot in the alfalfa samples collected in Gansu Province. This is the first report of M. tabacinum being a pathogen of alfalfa in China.

An arbuscular mycorrhizal fungus and Epichloë festucae var. lolii reduce Bipolaris sorokiniana disease incidence and improve perennial ryegrass growth

Leaf spot of perennial ryegrass (Lolium perenne) caused by Bipolaris sorokiniana is an important disease in temperate regions of the world. We designed this experiment to test for the combined effects of the arbuscular mycorrhizal (AM) fungus Claroideoglomus etunicatum and the grass endophyte fungus Epichloë festucae var. lolii on growth and disease occurrence in perennial ryegrass. The results show that C. etunicatum increased plant P uptake and total dry weight and that this beneficial effect was slightly enhanced when in association with the grass endophyte. The presence in plants of both the endophyte and B. sorokiniana decreased AM fungal colonization. Plants inoculated with B. sorokiniana showed the typical leaf spot symptoms 2xa0weeks after inoculation and the lowest disease incidence was with plants that were host to both C. etunicatum and E. festucae var. lolii. Plants with these two fungi had much higher activity of peroxidases (POD), superoxide dismutase (SOD) and catalase (CAT) and lower values of malondialdehyde (MDA) and hydrogen peroxide (H2O2). The AM fungus C. etunicatum and the grass endophyte fungus E. festucae var. lolii have the potential to promote perennial ryegrass growth and resistance to B. sorokiniana leaf spot.

First report of Septoria apocyni causing spot blight on the species of Apocynum venetum and Poacynum pictum in China.

The species of Apocynum venetum and Poacynum pictum grow widely from the middle to northwestern regions of China. During the summers of 2011 to 2013, a spot blight was found in wild and cultivated both species in Altay Prefecture of the Xinjiang Uygur Autonomous Region, China. The spot blight caused leaf yellowing and leaf drop, and serious damage to plant phloem. Lesions were circular to irregular, and the diameter of lesions on A. venetum and P. pictum was 1.84 to 6.84 × 1.23 to 4.24 mm and 2.05 to 7.09 × 1.46 to 5.65 mm, respectively. Pycnidia were 70 to 115 × 52 to 120 μm, scattered, spherical, buried, and had a brown hard shell with a prominent ostiole. Conidia were colorless, needle-shaped, or linear. The conidia base was obtuse, containing 3 to 5 indistinct septa, 46.3 to 110.3 × 2 to 2.5 μm. Fungal cultures were obtained by cutting 1-cm-long infected leaf pieces from the margins of the lesions following routine surface sterilizing procedures. The sections were placed on potato dextrose agar (PDA) in petri dishes and incubated at 23°C for 4 weeks (4). Hyphae had septa, the aerial and base mycelium was white and rufous, and the back of the colony was sunken and cracked after 2 weeks, but no spore was observed. To verify the identity, total DNA was extracted directly from fungal mycelium with a UNIQ-10 fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China) and PCR amplification performed with primers ITS1/ITS4 (3). A 512-bp PCR product was sequenced and contrasted with GenBank sequences using BLAST, which revealed 99% identity with Septoria sp. (GenBank Accession No. KC134322.1). To confirm pathogenicity, A. venetum and P. pictum were planted in pots and grown in a greenhouse. After 6 weeks of growth, plants were inoculated by spraying a mycelial suspension onto the foliage while control plants received a similar application of sterilized distilled water. Five pots (3 plants per pot) were used for each treatment. The pots were then placed on plates filled with tap water and covered with Plexiglas hoods in the greenhouse at 20 to 25°C. Lesions began to appear 6 to 7 days after inoculation with the mycelial suspension, whereas control plants remained healthy. The average disease incidence was 19.3%. The symptoms and morphology were similar to Septoria apocyni in Teterevnikova (2). It was determined that spot blight of A. venetum and P. pictum was caused by S. apocyni based on morphological comparison. There is one relevant literature report of spot blight on A. venetum and P. pictum in China, but without any details of the pathogenicity or morphology of the pathogen (1). We believe that this is the first report of S. apocyni occurring on the species of A. venetum and P. pictum in China. References: (1) W. Sun et al. Special Economic Animal and Plant 8:23, 2005. (2) D. N. Teterevnikova. Page 79 in: Septoria sp. Fungus of USSR. Armenian Academy of Sciences Publishing, Armenia, USSR, 1987. (3) G. J. M. Verkley et al. Mycologia 96:558, 2004. (4) W. Zhang et al. Plant Dis. 96:1374, 2012.

The occurrence of rust disease, and biochemical and physiological responses on Apocynum venetum plants grown at four soil water contents, following inoculation with Melampsora apocyni

We studied the development of rust disease, and biochemical and physiological responses, on Apocynum venetum plants inoculation with Melampsora apocyni that were growing in a greenhouse at four relative soil water contents. The soil conditions were 25% (severe drought), 50% (mild drought), 75% (optimal) and 100% (waterlogging) relative soil water content. Plants exposed to drought and waterlogging stress had a lower number of open stomata before inoculation, corresponding with the disease index on the 10th day after inoculation being lower than that of the optimal soil water condition. Inoculated plants exposed to severe and mild drought stress had a gradually enhanced resistance to the rust disease from the 10th day after inoculation, corresponding with the enhanced activity of polyphenol oxidase and phenylalanine ammonialyase. For the inoculated plants exposed to severe drought stress, hydrogen peroxide always remained at the highest level for any treatment, and they had a rapidly enhanced activity of peroxidase, two factors that were associated with suppression of disease development. A. venetum plants exposed to double stress of waterlogging and disease had a high activity of peroxidase that not only removed reactive oxygen to prevent or reduce cell injury but also enhanced resistance to the rust disease. In addition, a rapidly enhanced activity of phenylalanine ammonialyase in the waterlogging condition from the 25th day after inoculation was also associated with an enhanced resistance to the rust disease. Drought and waterlogging stress had a negative effect on the leaf photosystem, and in particular, there was a significant decrease in the net photosynthetic rate with an increase in the duration and degree of drought stress, and this lead to a statistically significant decrease in the weight of aboveground tissue compared with that of plants under optimal soil water condition (Pxa0<xa00.05). Inoculating with M. apocyni had a slight effect on photosynthesis of plants during early disease development, but the physiological function of diseased leaves under the drought stress was damaged more seriously than that of non-inoculated plants in later disease development, leading to a large reduction in the net photosynthetic rate. However, this reduction did not cause a statistically significant (Pxa0>xa00.05) decrease in the weight of aboveground tissue compared with that of non-inoculated plants under drought stress.

Arbuscular mycorrhiza fungi increased the susceptibility of Astragalus adsurgens to powdery mildew caused by Erysiphe pisi

ABSTRACT Powdery mildew caused by Erysiphe pisi is a major factor that affects the growth of standing milkvetch (Astragalus adsurgens). As arbuscular mycorrhizal fungi (AMF) have shown to be enhancing the resistance of plants to biotrophic pathogens such as powdery mildew, a study was carried out to look at the effects of three AMF, either singularly or in combination, on the growth of standing milkvetch and susceptibility to E. pisi. The results showed that the presence of AMF enhanced the growth of standing milkvetch even though their presence in the roots increased susceptibility to this foliage pathogen compared with plants having no AMF. This increase in growth of plants with severe infection of powdery mildew was especially surprising as leaves contained lower levels of chlorophyll than plants without AMF and had a greater concentration of malondialdehyde, an indicator of the damage of cell membrane. The effects on the extent of growth and powdery mildew enhancement differed inconsistently with the type of AMF in roots. The effects on growth and powdery mildew were not related to intensity of AMF colonisation. The peroxidase (POD) was consistently higher activity (15% to 72%) in plants with AMF than plants without them.

Effects of dual inoculation of AMF and rhizobium on alfalfa (Medicago sativa) root rot caused by Microdochium tabacinum

This experiment aimed to test the combined effects of arbuscular mycorrhizal fungi (AMF) and rhizobium (Sinorhizobium medicae) on the alfalfa root rot (Microdochium tabacinum) disease. The results show a significant increase in alfalfa growth, induced by AMF, independent of inoculation with rhizobium. Inoculation with S. medicae increased alfalfa N concentration compared with un-inoculated plants. In the presence of both AMF and S. medicae, the N, P concentration of plant were significantly greater than in their absence. M. tabacinum caused plant branches wilt and significantly reduced plant total dry weight as well as N, P concentration. Inoculation of AMF increased nodule numbers, independent of the presence of other factors. AMF and rhizobium reduced alfalfa branches discoloration by 18%. Plant disease related enzyme, peroxidase (POD), and catalase (CAT) showed a positive response to both AMF and rhizobium, while the malondialdehyde (MDA) content responded negative.

Albinism and mosaicism in Apocynum venetum associated with viral infections in China

The viruses that cause the two symptoms, albinism and mosaic, of Apocynum venetum were identified by deep sequencing of small RNAs and confirmed using RT-PCR. The nucleotide sequence identities of the assembled contigs ranged from 84.77% to 96.58% when compared to the reference sequences of the five respective viral genomes in GenBank. Sequences of assembled contigs homologous to PiCV-1 accounted for 49.84% and 46.64% of the recovered viral sequences from the two infected A. venetum samples Ap-1 and Ap-2, respectively, which were the most abundant among all assembled virus sequences, followed by GLRaV-1, which accounted for 30.08% and 23.38%, respectively. Stealth virus, Tete virus, and Garlic virus were also present, in descending order of abundance, among the tested samples. The albinism symptom and mosaic symptom of A. venetum leaves was associated with infections by Pittosporum cryptic virus 1 (PiCV-1), Grapevine leafroll-associated virus 1 (GLRaV-1), Tete virus, Stealth virus and Garlic virus. This is the first report of A. venetum as a natural host of PiCV-1, GLRaV-1, and other viruses.

Bacillus subtilis subsp. spizizenii MB29 controls alfalfa root rot caused by Fusarium semitectum

Medicago sativa L. is the most important forage legume in China. Reducing production losses caused by disease is an essential aspect of maximising alfalfa production. In the current study a Fusarium semitectum isolate collected from alfalfa roots exhibiting symptoms of root rot was proven to infect alfalfa by fulfilling Kochs postulates. A bacterial strain, MB29, also collected from alfalfa roots, was evaluated as a potential biocontrol agent against F. semitectum and a range of other alfalfa pathogens using in vitro tests. It was found that MB29 reduced the mycelia growth of all the pathogens assessed, and in the case of F. semitectum by as much as 84.47%. Furthermore, in vivo test showed that MB29 reduced the severity of rot symptoms in alfalfa seedlings resulting from F. semitectum infection. Strain MB29 was subsequently classified as Bacillus subtilis subsp. spizizenii using the Biolog MicroLog microbial identification system and sequence analysis of its 16S rDNA gene. Taken together these results indicate that B. subtilis subsp. spizizenii MB29 has great potential for the control of root rot diseases in alfalfa.