Xiangli Dong

Effects of Temperature, Wetness Duration, and Moisture on the Conidial Germination, Infection, and Disease Incubation Period of Glomerella cingulata

Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emergent disease that results in severe defoliation and fruit spots. Currently, GLS is not effectively controlled in China due to a lack of understanding of its epidemiology. Therefore, the effects of temperature, wetness duration, and moisture on conidial germination, infection, and the disease incubation period of GLS were examined by inoculating cv. Gala apple leaves with a conidial suspension and performing in vitro germination assays. Conidia could germinate and form appressoria at temperatures ranging from 5 to 35°C, with an optimum temperature of 27.6°C. The germination of conidia required free water or a nearly saturated relative humidity, with only a few conidia germinating and forming appressoria when the RH was less than 99%. The conidial germination dynamics at 10, 25, and 30°C were well represented by three logistic models. The infection of cv. Gala apple leaves by conidia occurred at temperatures ranging from 15 to 35°C. The minimum wetness duration required for infection by conidia at different temperatures was described using a polynomial equation, and the lowest minimum wetness duration was 2.76 h, which occurred at 27.6°C according to the polynomial. Successful infection by conidia was represented by the number of lesions per leaf, which increased with extended wetness durations at the conidial infection stage for six tested temperatures, with the exception of 10°C, when the minimum wetness durations were satisfied. The associations of successfully infected conidia with wetness duration at temperatures of 15, 20, 25, and 30°C were described by four logistic models. Conidia infections developed into visible lesions at temperatures ranging from 15 to 30°C, and the shortest incubation period of 2 days was observed at 25°C. These data and models can be used to construct forecasting models and develop effective control systems for Glomerella leaf spot.

Toxins produced by Valsa mali var. mali and their relationship with pathogenicity.

Valsa mali var. mali (Vmm), the causal agent of apple tree canker disease, produces various toxic compounds, including protocatechuic acid, p-hydroxybenzoic acid, p-hydroxyacetophenone, 3-(p-hydroxyphenyl)propanoic acid and phloroglucinol. Here, we examined the relationship between toxin production and the pathogenicity of Vmm strains and determined their bioactivities in several assays, for further elucidating the pathogenesis mechanisms of Vmm and for developing new procedures to control this disease. The toxins were quantified with the high performance liquid chromatography (HPLC) method, and the results showed that the strain with attenuated virulence produced low levels of toxins with only three to four kinds of compounds being detectable. In contrast, higher amounts of toxins were produced by the more aggressive strain, and all five compounds were detected. This indicated a significant correlation between the pathogenicity of Vmm strains and their ability to produce toxins. However, this correlation only existed in planta, but not in vitro. During the infection of Vmm, protocatechuic acid was first detected at three days post inoculation (dpi), and the others at seven or 11 dpi. In addition, all compounds produced noticeable symptoms on host plants at concentrations of 2.5 to 40 mmol/L, with protocatechuic acid being the most effective compound, whereas 3-(p-hydroxyphenyl)propanoic acid or p-hydroxybenzoic acid were the most active compounds on non-host plants.

Agrobacterium tumefaciens-mediated transformation of Valsa mali: an efficient tool for random insertion mutagenesis.

Valsa mali is a causal agent of apple and pear trees canker disease, which is a destructive disease that causes serious economic losses in eastern Asia, especially in China. The lack of an efficient transformation system for Valsa mali retards its investigation, which poses difficulties to control the disease. In this research, a transformation system for this pathogen was established for the first time using A. tumefaciens-mediated transformation (ATMT), with the optimal transformation conditions as follows: 106/mL conidia suspension, cocultivation temperature 22°C, cocultivation time 72 hours, and 200 μM acetosyringone (AS) in the inductive medium. The average transformation efficiency was 1015.00 ± 37.35 transformants per 106 recipient conidia. Thirty transformants were randomly selected for further confirmation and the results showed the presence of T-DNA in all hygromycin B resistant transformants and also revealed random and single gene integration with genetic stability. Compared with wild-type strain, those transformants exhibited various differences in morphology, conidia production, and conidia germination ability. In addition, pathogenicity assays revealed that 14 transformants had mitigated pathogenicity, while one had enhanced infection ability. The results suggest that ATMT of V. mali is a useful tool to gain novel insight into this economically important pathogen at molecular levels.

Draft Genome Sequence of Botryosphaeria dothidea, the Pathogen of Apple Ring Rot.

ABSTRACT Botryosphaeria dothidea is a destructive pathogen infecting apple (Malus domestica) on the fruit and stem. Here, we present the draft genome sequence of B. dothidea (strain LW030101) using Illumina sequencing. The draft genome sequence provides useful information and acts as a platform for further research on the pathogen.

Induction of resistance mediated by an attenuated strain of Valsa mali var. mali using pathogen-apple callus interaction system.

To study the induced resistance in apple against Valsa mali var. mali (Vmm), a Vmm–apple callus interaction system was developed to evaluate the induced resistance of an attenuated Vmm strain LXS081501 against further infection by a virulent Vmm strain LXS080601. The infection index was up to 97.32 for apple calli inoculated with LXS080601 alone at 15 days after inoculation whereas it was only 41.84 for calli pretreated with LXS081501 followed by LXS080601 inoculation. In addition, the maximum levels of free proline, soluble sugar, and protein in calli treated with LXS081501 plus LXS080601 were 2.14 to 3.47 times higher than controls and 1.42 to 1.75 times higher than LXS080601 treatment. The activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and catalase (CAT) as well as β-1,3-glucanase and chitinase in apple calli inoculated with LXS080601 alone or LXS081501 plus LXS080601 increased significantly 24 hai and peaked from 48 to 120 hpi. However, in the latter treatment, the maximum enzyme activities were much higher and the activities always maintained much higher levels than control during the experimental period. These results suggested the roles of osmotic adjustment substances and defense-related enzymes in induced resistance.

Effects of Temperature, Humidity, and Wound Age on Valsa mali Infection of Apple Shoot Pruning Wounds

Valsa canker, caused by Valsa mali, is a destructive disease of apple in China. The pathogen infects apple branches, mainly through pruning wounds, and causes branch and tree death. To determine the conditions required for V. mali infection through pruning wounds and growth within the xylem, pruning wounds on 1- to 4-year-old apple branches were inoculated with conidia in vitro under artificially controlled conditions and in vivo in the orchard. The effects of temperature, wetness duration, and wound age on conidial infection through pruning wounds as well as hyphal growth in the xylem were examined. The results showed that, after invading through pruning wounds, V. mali hyphae grew along xylem vessels, tracheids, and rays, expanding longitudinally and laterally. The hyphae could enter adjacent xylem vessels and tracheids through micropores to form a dense hyphal network. Wetness duration did not exhibit an essential effect on conidial infection from pruning wounds. Conidia spread to pruning wounds with rainwater could infect the xylem without any other extra moisture. Temperature for V. mali conidia infection through pruning wounds and hyphal extension in the xylem ranged from 5 to 35°C, with the optimum at 20°C. Pruning wounds made in late March were susceptible to V. mali infection in March, April, and May; the susceptibility was markedly deceased by June, and the pathogen could barely infect through the pruning wounds in November. The infected pruning wounds began to show symptoms from the spring of the following year. More than half of the observed Valsa canker lesions emerged in the spring of the second year, and new canker twigs were also developed from the inoculations in the spring of the third year. March, April, and May are the critical periods for protecting pruning wounds against infection by V. mali in China, and coating pruning wounds with protective film immediately after pruning is an easy and effective measure to protect the pruning wounds.

Analysis of the dissipation kinetics of thiophanate‐methyl and its metabolite carbendazim in apple leaves using a modified QuEChERS–UPLC–MS/MS method

As one of the main fungicides for the apple leaf disease control, thiophanate-methyl (TM) mainly exerts its fungicidal activity in the form of its metabolite carbendazim (MBC), whose dissipation kinetics is very distinct from that of its parent but has been paid little attention. The aim of this work was to investigate the dissipation kinetics of TM and its active metabolite MBC in apple leaves using a modified QuEChERS-UPLC-MS/MS method. The results showed that TM and MBC could be quickly extracted by this modified QuEChERS procedure with recoveries of 81.7-96.5%. The method linearity was in the range of 0.01-50.0 mg kg-1 with the quantification limit of 0.01 mg kg-1 . Then this method was applied to the analysis of fungicide dissipation kinetics in apple leaves. The results showed that the dissipation kinetics of TM for the test in 3 months can be described by a first-order kinetics model with a DT50 (dissipation half-life) range of 5.23-6.03 days and the kinetics for MBC can be described by a first-order absorption-dissipation model with the Tmax (time needed to reach peak concentration) range of 4.78-7.09 days. These models can scientifically describe the behavior of TM and MBC in apple leaves, which provides necessary data for scientific application.