Fruit Ring Rot Research Articles

Melatonin enhances resistance to Botryosphaeria dothidea in pear by promoting jasmonic acid and phlorizin biosynthesis

Ring rot, caused by Botryosphaeria dothidea, is an important fungal disease of pear fruit during postharvest storage. Melatonin, as a plant growth regulator, plays an important role in enhancing the stress resistance of pear fruits. It enhances the resistance of pear fruits to ring rot by enhancing their antioxidant capacity. However, the underlying mechanism remains unclear. In this study, we examined the effect of melatonin on the growth of B. dothidea. Results showed that melatonin did not limit the growth of B. dothidea during in vitro culture. However, metabolomics and transcriptomics analyses of ‘Whangkeumbae’ pear (Pyrus pyrifolia) revealed that melatonin increased the activity of antioxidant enzymes, including peroxidase (POD), superoxide dismutase (SOD), and polyphenol oxidase (PPO), in the fruit and activated the phenylpropanoid metabolic pathway to improve fruit resistance. Furthermore, melatonin treatment significantly increased the contents of jasmonic acid and phlorizin in pear fruit, both of which could improve disease resistance. Jasmonic acid regulates melatonin synthesis and can also promote phlorizin synthesis, ultimately improving the resistance of pear fruit to ring rot. In summary, the interaction between melatonin and jasmonic acid and phlorizin enhances the antioxidant defense response and phenylpropanoid metabolism pathway of pear fruit, thereby enhancing the resistance of pear fruit to ring rot disease. Our results provide new insights into the application of melatonin in the resistance to pear fruit ring rot.

Label-free Raman microspectroscopic imaging with chemometrics for cellular investigation of apple ring rot and nondestructive early recognition using near-infrared reflection spectroscopy with machine learning

Apple ring rot caused by Botryosphaeria dothidea can cause fruit decay during the growth and storage stages of apple fruit. Understanding the infection process and cellular defense response at the cellular micro-level holds immense importance in the field of prevention and control. Consequently, there is a pressing need to develop suitable chemical imaging analysis methods. Here we proposed a label-free, high-throughput imaging method for cellular investigation of apple fruit ring rot infected by Botryosphaeria dothidea, based on confocal Raman microspectroscopic imaging technology combined with multivariate curve resolution-alternating least squares algorithm (MCR-ALS). We conducted Raman measurements on every apple fruit and obtain an image cube. This cube was then unfolded into an augmented matrix in a column-wise manner. We proceeded with simultaneous MCR-ALS analysis, resolving the single-substance spectrum and concentration profile from the mixed signals. Lastly, the accurate and pure molecular imaging of low methoxyl pectin, high methoxyl pectin, cellulose, lignin, and phenols were realized by refolding the resolved concentration data to construct the composition image. Thereafter, we realized the study of the spatial-temporal changes distribution of the above substances in the cuticle and cell wall of green and red apples at different stages of infection. The imaging method proposed in this paper is expected to provide a chemical imaging strategy for studying pathogen infection process and fruit defense response at the cellular level. In addition, by utilizing a fiber-optic probe near-infrared reflection spectrometer in conjunction with machine learning, we developed a rapid and non-destructive classification method. This method allows for the timely identification of apples exhibiting early infection by Botryosphaeria dothidea. Notably, both principal component analysis-quadratic discriminant analysis and support vector machine achieved a classification accuracy of 100%.

Involvement of the autophagy-related gene BdATG8 in development and pathogenicity in Botryosphaeria dothidea

Botryosphaeria dothidea is a destructive fungal pathogen that causes Botryosphaeria canker and fruit ring rot on apple worldwide. Autophagy is a process of self-degradation that maintains intracellular homeostasis via lysosomal pathway. To date, the biological role of autophagy in B. dothidea remains unknown. In this study, we identified and characterized the autophagy-related gene BdATG8 in B. dothidea. BdATG8 was able to restore the defect in nitrogen starvation tolerance of Saccharomyces cerevisiae ATG8 deletion mutant. GFP-BdAtg8 was shown to be a useful marker for monitoring autophagy in B. dothidea. Target deletion of BdATG8 (ΔBdAtg8) blocked autophagy and significantly impaired mycelial growth, conidiation and perithecium formation. In addition, ΔBdAtg8 showed significantly increased sensitivity to phytoalexin and oxidative stress, suggesting that BdATG8 plays critical roles in overcoming phytoalexin and reactive oxygen species (ROS)-mediated plant immunity. Pathogenicity assays revealed that ΔBdAtg8 almost lost ability to infect hosts. Overall, our results indicate that BdATG8 plays an important role in fungal development, stress responses and pathogenesis in B. dothidea.

Genome Assembly and Annotation of Botryosphaeria dothidea sdau11-99, a Latent Pathogen of Apple Fruit Ring Rot in China.

Botryosphaeria dothidea is a latent and important fungal pathogen on a wide range of woody plants. Fruit ring rot caused by B. dothidea is a major disease in China on apple. This study establishes a high-quality, nearly complete, and well-annotated genome sequence of B. dothidea strain sdau11-99. The findings of this research provide a reference genome resource for further research on the apple fruit ring rot pathogen on apple and other hosts.

Biocontrol efficiency of Meyerozyma guilliermondii Y-1 against apple postharvest decay caused by Botryosphaeria dothidea and the possible mechanisms of action

Apple ring rot, caused by Botryosphaeria dothidea, is one of the important diseases in China. This pathogen infects branches and fruit and also results in fruit decay during storage. Biocontrol agents have been proposed to reduce apple decays during storage and are considered as a promising alternative strategy to traditional chemical treatment. In this study, Meyerozyma guilliermondii Y-1, isolated from healthy grape fruit, was firstly evaluated for its biocontrol efficiency against B. dothidea in postharvest apple fruit, and the possible mechanisms were investigated. The results revealed that M. guilliermondii Y-1 treatment effectively reduced apple ring rot caused by B. dothidea in vivo. The disease incidence and lesion diameter were reduced by 32.22% and 57.51% compared with those of control fruit. Furthermore, the use of filtrate and autoclaved culture of M. guilliermondii Y-1 also showed a certain degree of control efficiency against fruit ring rot. M. guilliermondii Y-1 significantly inhibited the mycelial growth and spore generation of B. dothidea in vitro and exhibited an obvious ability to colonize in apple fruit wounds and surface at 25 °C or 4 °C. In addition, M. guilliermondii Y-1 treatment significantly enhanced the activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonialyase (PAL), and polyphenoloxidase (PPO), promoted the total phenolics content, and alleviated lipid peroxidation in apple fruit. As expected, we found that the expression of four pathogenesis-related proteins genes (MdPR1, MdPR5, MdGLU, and MdCHI) was remarkably increased by M. guilliermondii Y-1 treatment. Our data together suggest that M. guilliermondii Y-1 is a potential biocontrol agent against B. dothidea postharvest infection in apple fruit, partially through inhibiting mycelial growth and spore germination of B. dothidea, competing for space and nutrient with pathogen, and inducing resistance in apple fruit by stimulating a series of defense responses.

Mapping Gene Markers for Apple Fruit Ring Rot Disease Resistance Using a Multi-omics Approach.

Apple fruit ring rot (FRR), caused by Botryosphaeria dothidea, is a worldwide disease that impacts Asian apple production regions. However, no substantial progress has thus far been made toward the mapping of candidate genes or the development of effective genetic makers. In this five-year study, the resistance of 1,733 F1 hybrids from the cross ‘Jonathan’ × ‘Golden Delicious’ was phenotyped by non-wounding inoculation with four B. dothidea isolates. We first conducted systematic comparison of different analytic strategies for bulk segregant analysis by re-sequencing (BSA-Seq) and obtained suitable one for outbreeding species such as Malus. Forty-six quantitative trait loci (QTL) for resistance/susceptibility to the four isolates, including one QTL ‘hotspot’ on chromosome 14, were identified via BSA-Seq. Using integrated multi-omics strategies including RNA-sequencing, parental re-sequencing, BSA-Seq and meta-analysis of RNA-sequencing, fifty-seven candidate genes and corresponding functional mutations from the QTL were predicted. Functional mutations located on the candidate genes were validated using kompetitive allele-specific PCR in hybrids and Malus germplasm accessions with extremely resistant/susceptible phenotypes. Ten effective markers for apple ring rot were developed. The results provide an example of rapid candidate gene mapping for complex traits in outbreeding species.

Genetic Mapping Reveals Sophisticated Responses of Malus domestica to Botryosphaeria dothidea Isolates

Infection by the globally distributed pathogenic fungus Botryosphaeria dothidea (Moug.) Ces. et de Not. causes bot canker on the stems, branches and limbs or causes fruit ring rot (FRR) on the fruit in apple. To investigate the relationship between resistance to bot canker and FRR and among resistance mechanisms in response to different pathogen isolates, 34 major gene loci and six quantitative trait loci (QTLs) for bot canker and FRR resistance/susceptibility were genetically mapped on Malus genome using an F1 hybrid population (Jonathan × Golden Delicious) in 2008 and 2009. None of the QTLs for bot canker and FRR overlapped with the region of the major gene loci. Some of the FRR- and bot canker-associated loci either coincided or overlapped, and some were independent, suggesting that the responses of different organs to the pathogen would be correlated but might deviate from each other. Some major gene loci and QTLs associated with different pathogen isolates of bot canker or, FRR were mapped to different regions of the genome. The results indicated that not only did the resistance mechanisms differ between bot canker and FRR but also that genetic differentiation occurred among pathogen isolates.

Inheritance of and Molecular Markers for Susceptibility of Malus domestica to Fruit Ring Rot (Botryosphaeria dothidea)

AbstractTo analyse the inheritance of fruit ring rot (FRR) resistance and to screen for microsatellite markers linked to resistance/susceptibility, 875 apple hybrid seedlings (Malus domestica, Jonathan × Golden Delicious) were inoculated with five isolates of Botryosphaeria dothidea in 2 years (2008 and 2009). The results indicated that incidence and non‐incidence were qualitatively segregated, and incidence was dominant to non‐incidence. The variation in susceptibility within this population was attributed to the segregation of three major genes. For the phenotype of incidence, the severity of lesion development was a quantitative trait. From 230 published microsatellite primer pairs, six markers were identified that were linked to the susceptibility to FRR. CH04d02‐120 and Hi08g12‐190, located in LG12 and LG2, respectively, were linked to susceptibility to the pathogen isolate Mx1, and their map distances to the susceptibility loci were 8.2 and 5.1 centimorgan (cM), respectively. CH01e01‐120 and CH02c02b‐100, which were linked to susceptibility to Ls1, were located in LG14 and LG4, and the map distances to the susceptibility loci were 16.9 and 8.4 cM, respectively. CH05d11‐150 and CH03a03‐230, linked to susceptibility to Lw048, were located in LG12 and LG14; for both of them, the map distance was 13.4 cM.

First Report of Gummosis Disease of Japanese Apricot Caused by Botryosphaeria dothidea in Taiwan.

Japanese apricot (Prunus mume Sieb. et Zucc.) is an economically important fruit crop grown on more than 10,000 ha in Taiwan. During May 2008, twigs of Japanese apricot trees in the commercial farms of Renai Region (Nantou County) showed symptoms of gummosis disease, with 12 to 18% of the trees affected. The disease was more severe on trees weakened by drought stress. Limb and twig infections began around lenticles as small, sunken, discolored lesions at the margins of wounds. Following infection, cortical cells collapsed, bark became depressed, and blisters developed, which were often cracked with whitish gummy exudation. Necrotic areas were seen on the cortical tissues. Leaves showed yellowing and drooping. In winter months, numerous black pycnidia or perithecia formed on infected twigs. Single conidial isolates of the pathogen were obtained from diseased twigs on acidified potato dextrose agar (PDA) incubated at 25 ± 1°C for 3 days. On the basis of morphological characteristics, the fungus was identified as Botryosphaeria dothidea (3). Conidia (17 to 22.6 × 4.3 to 6.0 μm) were hyaline, unicellular, and spindle shaped. Asci (78 to 125 × 15 to 17 μm) were hyaline, bitunicate, clavate, and eight spored. Ascospores (18 to 22 × 7.0 to 8.2 μm) were hyaline and spindle shaped or fusoid. The pathogen identity was further confirmed by PCR amplification and sequencing of ribosomal DNA internal transcribed spacer from the fungus with the primers ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (4), and a representative sequence was deposited in NCBI GenBank (Accession No. GU594225). The sequence showed 99 to 100% homology with previously characterized strains of B. dothidea (GenBank Accession Nos. EU441944, DQ177876, and AY786320). Pathogenicity tests were conducted with inoculum prepared by culturing the fungus on PDA under a continuous photoperiod of 128 ± 25 μE·m-2·s-1 at 25°C for 3 days. Shallow cuts (3 × 3 × 3 mm) were made on 12- to 15-month-old healthy twigs with a scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of conidial suspension (105 conidia/ml) of the fungus. Two twigs on each of six trees were inoculated. Inoculated areas were covered with moist, sterile cotton and the entire twigs were enclosed in plastic bags. Twigs were inoculated with 5-mm PDA discs or sterile water for controls. The symptoms described above were observed on all inoculated twigs 14 days after inoculation, whereas control twigs remained healthy. Reisolation from the inoculated twigs consistently yielded B. dothidea. In Taiwan, B. dothidea has been reported as the causal agent of gummosis of peach (1) and fruit ring rot of pear (2); however, to our knowledge, this is the first report of B. dothidea causing gummosis on Japanese apricot.

First Report of Gummosis Disease of Plum (Prunus salicina) Caused by a Botryosphaeria sp. in Taiwan.

Plum (Prunus salicina Lindell) is grown on more than 3,870 ha in Taiwan. In 2004, a gummosis disease was observed on plum in the Ming Jian Region of Nantou County (120.675°E, 23.919°N), with 15% of the trees affected. Infections started on the current year's growth, primarily through lenticels, and formed small, sunken, discolored lesions. At later stages, white gum exuded from the lesions. Circular to oval, brown, necrotic areas were seen on the inner bark. Severely infected twigs showed defoliation and dieback. During the winter months, numerous black pycnidia or perithecia formed on infected twigs. Single conidial isolates of the pathogen were obtained from diseased twigs on acidified potato dextrose agar (PDA) and incubated at 25 ± 1°C for 3 days. On the basis of morphological traits, the fungus was identified as a Botryosphaeria sp. according to the CMI descriptions of Botryosphaeria ribis (3). Conidia (14.2 to 26.8 × 4.3 to 7.2 μm) were single celled, hyaline, and spindle shaped. Asci (105 to 135 × 12.5 to 15.5 μm) were hyaline, clavate, and bitunicate. Ascospores (18 to 22 × 7.0 to 8.2 μm) were hyaline and spindle shaped or fusoid. For pathogenicity tests, inoculum was prepared by culturing the fungus on PDA under continuous fluorescent light (128 ± 25 μE·m-2·s-1) at 25°C for 3 days. Two twigs on each of six trees were inoculated. Sharp incisions (3 × 3 × 3 mm) were made on healthy twigs (12 to 15 months old) with a sterilized scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of a conidial suspension (105 conidia/ml) of the fungus. Inoculated areas were covered with moist, sterile cotton and the entire twigs were enclosed in plastic bags. Twigs inoculated with 5-mm PDA discs or sterile water alone served as controls. The symptoms described above were observed on all inoculated twigs 14 days after inoculation, whereas control twigs did not develop any disease symptoms. Reisolation from the inoculated twigs consistently yielded the Botryosphaeria sp., thus fulfilling Koch's postulates. Botryosphaeria spp. have been reported to cause stem blight of many plants in temperate and tropical regions of the world (4). In Taiwan, B. dothidea has been reported as the causal agent of gummosis disease of peach (1) and fruit ring rot of pear (2); however, to our knowledge, this is the first report of a Botryosphaeria sp. causing gummosis of plum.