Hazelnut husk brown rot has been identified as a new disease in Liaoning Province in recent years. The objective of this study as to identify the pathogen. [Method] In this study, a standard sample of hazelnut husk brown rot was collected from Songmudao Base in Dalian City, Liaoning Province. The pathogen was identified by the studies of the morphology, pathogenicity, and analyses of ITS and LSU sequences. The pathogen was isolated and purified, which was confirmed by Koch’s postulates. The symptoms after inoculation were the same as those collected directly from a diseased tree, which showed that it was the pathogenic fungus. The cultural characteristics and conidia and the morphology of the pathogenic fungi were similar to those of
Hazel (
China is one of the centers of origin of hazelnuts. Hazelnut is rich in plant resources, and Chinese citizens have a long history of eating these prized nuts. They are grown on approximately 1.3 million hm2. There is approximately 1.3 million hm2 Ping hazelnut (
With the nutritional and medicinal value of hazelnuts gradually gaining attention, the area planted to this crop has undergone a rapid increase. Unfortunately, this growth has been followed by a serious occurrence of disease. In view of this, this study reported and systematically identified the new hazelnut husk brown rot disease in Liaoning Province to provide a further theoretical basis for the cultivation and management, in-depth research and prevention of this disease.
The study found that the Hazelnut husk brown rot has occurred seriously in Northeast China in recent years. It has been identified in Jinzhou County, Dalian City, Heishan County, Jinzhou City and Huanren County, Benxi City, and it has spread rapidly. In severe cases, 50%–60% of the husks of the whole tree are browned and decayed, which seriously affects the yield.
Few official reports have been made. Sezer
The Hazelnut husk brown rot samples were collected from Songmudao Base (121°75'E,39°40'N) in Dalian City, Liaoning Province, China during July 2019 to July 2020. The hazel tree species was Ping’ou hybrid hazelnut “Yuzhui” in five orchards. The characteristics of the symptoms of the disease was recorded and followed up in the field.
Observation of the morphological characteristics of the pathogen: The pathogen was isolated and purified by conventional isolation methods (
Following Koch’s rule, five-year-old growing hazelnut plants in the orchard were selected, and 20 hazelnut husks were inoculated with fungal plates. Ten stitches with a sterilized needle were made on the hazelnut husks. Plates that were 7 mm across were rubbed with a sterile spreader. The mycelial surfaces were covered toward where the hazelnut husks were pricked and incubated under humid conditions for 36 h. The pathogenic results were observed. A conventional tissue isolation method was used to separate the pathogen, and the pathogen was observed under a microscope after cultivation for identification.
Denaturation: The mycelia in the PDA media were picked and denatured in a 50 μL kit (TaKaRa Lysis Buffer for Microorganism to Direct PCR, Code No. 9164, Dalian, China), and the supernatant was centrifuged to obtain the template. PCR amplification: The target fragment was amplified using a kit (TaKaRa Fungi Identification PCR Kit, Code No. RR178). The ITS amplified primers were ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC). The LSU amplified primers were LR0R (5’-ACCCGCTGAACTTAAGC-3’) and LR7 (5’-TACTACCACCAAGATCT-3’). The PCR product was purified using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit v. 4.0 (Code No. 9762) cutting edge fragments for recovery. The DNA was also sequenced, and the results were aligned with those of the NCBI. These were completed by Takara Biotechnology Company.
The harvested tissue was blocked and fixed: Targeted fresh tissues should be selected to minimize mechanical damage, such as pulling, contusion and extrusion. A sharp blade was used to cut and quickly harvest fresh tissue blocks within 1–3 min. The area of tissue block should be no more than 3 mm2. The washed tissue blocks were immediately fixed by electron microscopy fixative for 2 h at room temperature, and then transferred to 4°C for preservation and transportation. Post-fix: The tissue blocks were washed with 0.1 M phosphate buffer (PB) (pH 7.4) three times, 15 min each. The tissue blocks were then transferred into 1% OsO4 in 0.1 M PB (pH 7.4) for 1–2 h at room temperature. After that, the tissue blocks were washed in 0.1 M PB (pH 7.4) three times, 15 min each. The samples were dehydrated as follows: 30% ethanol for 15 min; 50% ethanol for 15 min; 70% ethanol for 15 min; 80% ethanol for 15 min; 90% ethanol for 15 min; 95% ethanol for 15 min; and two changes of 100% ethanol for 15 min. Finally, the blocks were treated with isoamyl acetate for 15 min. Drying: The samples were dried with a Critical Point Dryer. Conductive metal coating: The specimens were attached to metallic stubs using carbon stickers and sputter-coated with gold for 30 s. The specimens were observed and photographed with a scanning electron microscope.
The pathogen primarily harmed the husks of hazelnut trees, causing husk browning. High humidity caused many husks to turn brown and rot, seriously affecting hazelnut production. Hazelnut husk brown rot occurred on mature husks in early July. As the husks gradually developed, the pathogen first infected the middle of the husk. At first, they formed light brown oval disease spots. As the husks gradually grew larger, disease spots also gradually expanded to the edge of husks. The color of the diseased spots deepened, and the color on the edge of the spots became deeper than the center of the spots, which became dark brown. The disease spots gradually expanded into pieces. Subsequently, the pathogen gradually infected the nutshells, thus, hindering normal kernel growth and affecting the production of hazelnuts during July to August. When the humidity was high to 75%, a gray-black mildew layer occurred on the husks. When the conditions were suitable, the leaves around the sick husks were also affected, and the leaves became browned and withered.
The pathogens on PDA medium produced mycelia that were grey and white in the early stage and grey and flocculant after old ripening (
Compared with the symptoms under natural conditions, the color of the artificially inoculated hazelnut husks was slightly darker, and the speed of onset and plaque expansion took place more quickly. After inoculation, the symptoms were consistent with those under natural conditions in the field (
PCR amplification was performed for the measured strains, and electrophoresis revealed fragments of approximately 490 bp in size (
PCR amplification was performed for the measured strains, and electrophoresis revealed fragments of approximately 1311 bp in size (
The relevant data and results of identification showed that the isolated pathogen caused by hazelnut husk brown rot collected in this test was classified as Fungi Imperfecti, Hyphomycetes, Moniliales, Moniliaceae,
Scanning electron microscopy (SEM) observations of
Species of
Few official reports have been made for hazelnut husk disease. The prevalence of
During repeated recycling in the trials, the pathogen was found to be highly susceptible to hazelnut husks through wounds. The trees were more susceptible to infection in warm, rainy and foggy environments, which facilitated the spread of the pathogen. The trial results indicate that the symptoms of inoculation were the same as those under natural conditions. This study on the new disease of hazelnut still merits study on the biological characteristics of the pathogen. The host range of the pathogen needs to be thoroughly studied, as do the characteristics of its infection process and its mechanism, to provide a systematic theoretical basis for disease control in production.