The miR528-D3 Module Regulates Plant Height in Rice by Modulating the Gibberellin and Abscisic Acid Metabolisms.

Plant height, as one of the important agronomic traits of rice, is closely related to yield. In recent years, plant height-related genes have been characterized and identified, among which the DWARF3 (D3) gene is one of the target genes of miR528, and regulates rice plant height and tillering mainly by affecting strigolactone (SL) signal transduction. However, it remains unknown whether the miR528 and D3 interaction functions in controlling plant height, and the underlying regulatory mechanism in rice. In this study, we found that the plant height, internode length, and cell length of internodes of d3 mutants and miR528-overexpressing (OE-miR528) lines were greatly shorter than WT, D3-overexpressing (OE-D3), and miR528 target mimicry (OE-MIM528) transgenic plants. Knockout of D3 gene (d3 mutants) or miR528-overexpressing (OE-miR528) triggers a substantial reduction of gibberellin (GA) content, but a significant increase of abscisic acid (ABA) accumulation than in WT. The d3 and OE-miR528 transgenic plants were much more sensitive to GA, but less sensitive to ABA than WT. Moreover, the expression level of GA biosynthesis-related key genes, including OsCPS1, OsCPS2, OsKO2 and OsKAO was remarkably higher in OE-D3 plants, while the NECD2 expression, a key gene involved in ABA biosynthesis, was significantly higher in d3 mutants than in WT and OE-D3 plants. The results indicate that the miR528-D3 module negatively regulates plant height in rice by modulating the GA and ABA homeostasis, thereby further affecting the elongation of internodes, and resulting in lower plant height, which adds a new regulatory role to the D3-mediated plant height controlling in rice.

First Report of Leaf Spot Caused by Colletotrichum fructicola on Kiwifruit in China.

Kiwifruit (Actinidia spp.) is an important fruit with high nutritional and economic value, which is widely cultivated in China. In April 2021, leaf spots were observed on the leaves of 'Xuxiang' (A. deliciosa) in a kiwifruit plantation of Hefei city, Anhui province, China (117°26'E, 31°85'N). Disease incidence was about 10% of the observed plants. Small yellow spots initially developed on the leaves and gradually expanded into irregular dark brown spots, and eventually the diseased leaves curled and withered. Leaf tissues (n=10, 5×5 mm) were collected from five infected plants, sterilized in 75% ethanol solution for 30 s and 1% NaOCl for 5 min, washed, dried and plated on PDA at 25°C. In total, ten isolates were obtained, including two previously reported Botryosphaeria dothidea (Zhou et al. 2015) and Diaporthe actinidiae strains (Bai et al. 2017) and eight unknown isolates with similar morphology. All unknown isolates initially appeared white with many aerial hyphae, and at the later stage, the center of all colonies turned gray. Colonies were transferred to new PDA with 0.1% yeast extract for three days. Then, aerial hyphae were scraped with sterile cotton swabs, and continued to grow for four days. Orange conidial masses were produced. Conidia were hyaline, smooth-walled, single-celled, cylindrical with broadly rounded ends, with average size around 4.1-5.5×13.2-18.2 µm (n=100). Appressoria (n=50) were ovoid in shape with average size around 4.9-6.7×8.6-11.8 µm. Morphological features were similar to Colletotrichum. gloeosporioides species complex (Weir et al. 2012). To confirm their species identification, internal transcribed spacers (ITS), ß-tubulin (TUB2), glyceraldehydec-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS) were amplified by PCR using the primer pairs ITS1/ITS4, Bt2a/Bt2b, GDF/GDR, ACT-512F/ACT-783R CHS-79F/CHS-234R, respectively (Weir et al. 2012). Based on alignment analysis, sequences of the eight unknown isolates were 100% homologous. The representative isolate LSD3-1 was selected for further study. BLAST analysis showed that the ITS (OM033371), TUB2 (OM044376), GAPDH (OM044377), ACT (OM044379) and CHS (OM044378) sequences of isolate LSD3-1 were 98.7%-100% identical with the collected sequences of C. fructicola strain ICMP:18581 (NR_144783, JX010405, JX010033, JX009866, JX009501). Phylogenetic analysis of multiple genes was conducted with the Maximum likelihood method using MEGA 7. Based on morphological and molecular characteristics, the LSD3-1 was identified as Colletotrichum fructicola (Prihastuti et al., 2009). Koch's postulates were performed on six one-year-old 'Xuxiang' plants, which were used to test pathogenicity in the greenhouse (at 28℃, relative humidity 80%, 16/8 h light/dark). Surface-sterilized leaves were sprayed with a conidial suspension (107 conidia/mL). Yellow and brown lesions were formed 14 to 21 days after inoculation, whereas the mock-inoculated controls remained asymptomatic. The experiment was performed three times. The fungus was reisolated and confirmed as C. fructicola by morphology and sequencing of all previously used genes. Although C. fructicola has been reported as a leaf spot disease on many plants (Shi et al. 2018), this is the first report of leaf spot caused by C. fructicola on kiwifruit in China. This result is helpful to better understanding the pathogen of kiwifruit leaf spot diseases in China and formulate effective control strategies.

First Report of flower rot Caused by Fusarium spp. on White kiwifruit in China.

As an important cultivated germplasm, cultivar 'White' (Actinidia eriantha) is appreciated by kiwifruit breeders because of its long shelf life, richness in ascorbic acid and peelable skin. In May 2020, about 1% to 3% of cultivated 'White' plants displayed typical symptoms of flower rot at farms in Hefei (117°25'E, 31°86'N) and Lujiang (117°27'E, 31°48'N), Anhui Province of China (Fig.1a&b). The infected flowers were yellowish at first, gradually turned brown, withered and shrunk, and finally died without blossoming. Infected flowers were surface sterilized in 70% alcohol for 30 s and 1% NaOCl for 3 min, then washed with double distilled water (ddH2O) for 5 times, and finally incubated in potato dextrose agar at 25 ± 2°C in the dark. Twenty fungal isolates were obtained and their colonies showed slightly raised center with dense and cotton-like mycelium. Colonies from Hefei (HF1~HF9) appeared pale yellow (Fig.1c), and those from Lujiang (LJ1~LJ11) showed purplish-red on PDA (Fig.1d). Two types of colonies grown on oatmeal agar were flat with few aerial hyphae (Fig.1e&f). On carnation leaf agar (CLA), isolate of HF1 produced abundant slightly curved macroconidia with 3 to 6 septa, 4.3-5.5×20.7-42.5 µm in size (n=100) (Fig.1g&h), without microconidia and chlamydospores observed. By contrast, macroconidia derived from LJ1 isolate were straight to slightly curved with 3 to 5 septa, 4.0-6.58×21.70-71.10 µm (n=100) in size (Fig.1i); Its chlamydospores were globose to subglobose (5.1 to 9.5 µm) on CLA (Fig.1j). Pathogenicity tests were performed on A. eriantha cv. 'White' flowers. The conidia suspension (105 spore/ml, 30 µL/flower) derived from the HF1 and LJ1 were separately dripped on flowers (n=100). Control flowers were treated with ddH2O. Two-week post-inoculation, all inoculated flowers were turned brown and withered (Fig.1k&m), whereas no symptoms were observed on the controls (Fig.1l&n). This experiment was repeated three times. All isolated and re-isolated pathogens from diseased flowers were subjected to molecular identification. Different molecular markers, including internal transcribed spacers (ITS), translation elongation factor (TEF-1α), calmodulin (CaM), RNA polymerase II subunit 1 (RPB1) gene and RNA polymerase II largest subunit (RPB2) gene, were amplified and sequenced to validate species identification (White et al. 1990; O'Donnell, et al. 1998; O'Donnell, et al. 2012). Based on sequence analysis, the re-isolated strains were identical to the inoculated individuals. Sequences of HF1 and HF2 or LJ1 and LJ2 were deposited in GenBank under accession numbers OK310710 to OK310713 (ITS), OK334291 to OK334294 (TEF-1α), OK412973 to OK412976 (CaM), OK412977 to OK412980 (RPB1), and OK484317 to OK484320 (RPB2), respectively. The BLAST search showed that the sequences of HF1 and HF2 showed 99 to 100% identity with ITS (NR_164594), TEF-1α (MK289601), CaM (MK289698), RPB1 (HM347158), and RPB2 (MK289754) of Fusarium luffae isolates. The sequences of LJ1 and LJ2 also revealed 99 to 100% identity with ITS (NR_121320), TEF-1α (AF212452), RPB1 (JX171459), and RPB2 (MW233412) of F. asiaticum isolates. F. asiaticum species-specific primers were used to detect LJ isolates (Yin et al. 2009), and the correct fragments were amplified (Fig.1o). Phylogenetic trees were constructed based on the tandem nucleotide sequences. Thus, both morphological and molecular criteria supported identification of HF group as F. Luffae (Fig.2a) and LJ group as F. asiaticum (Fig.2b). Fusarium spp. causing flower rot on many hosts have been previously reported (W. Elmer, et al. 2019; Liu, et al. 2021), but this is the first report of F. luffae and F. asiaticum on 'White' kiwifruit in China.

First Report of Root Rot on kiwifruits Caused by Athelia rolfsii in Hefei, China.

Kiwifruits (Actinidia ssp.), known as "King of vitamin C", have been wildly cultivated. In August 2020, about 15% of A. deliciosa (cv. Xuxiang) and A. macrosperma (rootstock) plants displayed symptoms typical of root rot at a farm in Hefei (117°25'E, 31°86'N), Anhui Province of China (Fig.1 a-b). Symptoms first appeared at the root and stem junction which were covered by cottony white mycelium during warm and humid summer. Then, the infected tissues were rotted, and subsequently the whole plant withered. Tan to brown sclerotia were observed on the basal stem epidermis and soil surface surrounding the stem (Fig.1 c-d). Infected plant tissues and sclerotia were collected for isolating the fungal pathogen. The samples were surface sterilized in 70% alcohol for 30 s, followed by 2% sodium hypochlorite for 3 min, washed five times with sterile double-distilled water (ddH2O), dried, placed on potato dextrose agar, and incubated at 25 °C in the dark. In total, twelve fungal isolates were obtained. The mycelia of all the isolates were white with a fluffy appearance (Fig.1 e). Sclerotia formed after 7 days were initially white (Fig.1 f) and gradually turned to dark brown (Fig.1 g) measuring 0.67 to 2.03 mm in diameter (mean = 1.367 ± 0.16 mm; n = 30). Hyphae were hyaline, septate. Some cells possessed multiple nuclei (Fig.1 h) and clamp connections (Fig.1 i). No spores were observed. For species-level identification, ITS1/ITS4 and EF1-983F/EF1-2218R primers were used to amplify the internal transcribed spacer regions (ITS) and translation elongation factor-1 alpha regions (TEF-1α), respectively (White et al. 1990; Rehner & Buckley 2005). Based on ITS and TEF-1α sequence analyses, all 12 isolates were categorized into two groups, group one including isolates NC-1 and NC-6~10 and group two containing NC-2~5 and NC-11~12. The length of ITS sequences for NC-1 (MW311079) was 684bp and 99% to 100% similar to Athelia rolfsii (MN610007.1, MN258360.1). Similarly, ITS sequences for NC-2 (MW311080) were 99% to 100% similar to A. rolfsii (MH858139.1; MN872304.1). Also, TEF-1α sequences of NC-1 (MW322687) and NC-2 (MW322688) were 96% to 99% similar to sequences of A. rolfsii (MN702794.1, GU187681.1, MN702789.1). Based on morphology and phylogenetic analyses (Fig.1 j&k), the isolates NC-1 and NC-2 were identified as Athelia rolfsii (anamorph Sclerotium rolfsii) (Mordue. 1974; Punja. 1985). To fulfill Koch's postulates, ten sclerotia of NC-1 were incorporated into the soil near stems of healthy Xuxiang plants (Fig.2 a). Similar treatments were also used for plants of A. macrosperma or A. arguta (Fig.2 g&m). Each control group had the same number of plants (n=3) for inoculating with ddH2O. The plants were kept in an incubator with a relative humidity of 80% and temperature of 28°C with 16/8 hours light/dark photoperiod. After twenty days, the pathogen-inoculated plants developed similar symptoms of root rot observed in the field (Fig.2 b-d, h-j, n-o). Similarly, four days after inoculation with sclerotia, leaves developed water-soaked lesions (Fig.2 e, k&p). No significant difference in pathogenicity was observed between NC-1 and NC-2. Non-inoculated control plants remained disease-free (Fig.2 f, l&q). The pathogenicity experiments were repeated three times. The pathogen was re-isolated from infected tissues and sclerotia, and isolates were confirmed as A. rolfsii by the ITS sequences. A. rolfsii has been reported to cause root rot in kiwifruit in the USA (Raabe. 1988). To our knowledge, this is the first report A. rolfsii causing root rot on kiwifruits in China.