Comparison on the Nutrient Plunder Capacity of Orychophragmus violaceus and Brassica napus L. Based on Electrophysiological Information

The nutrient metabolism, growth and development of plants are strongly affected by its nutrient plunder, and plants have different adaptive mechanisms to low-nutrient environments. The electrophysiological activities involve almost all life processes of plants. In this study, the active transport flow of nutrient (NAF) and nutrient plunder capacity (NPC) of plants were defined based on leaf intrinsic impedance (IZ), capacitive reactance (IXc), inductive reactance (IXL) and capacitance (IC) to evaluate the nutrient plunder capacity of plants for the first time. The results indicate that Orychophragmus violaceus had higher (p < 0.01) NPC and IC and lower (p < 0.01) IR, IXc, IXL and IZ as compared to Brassica napus L., which supports a superior ion affinity and that it could be better adapted to low-nutrient environments. UAF and NPC of plants exhibited good correlations with crude protein, crude ash and water content, and precisely revealed the plunder capacity and adaptive strategies of plants to nutrients. The present work highlights that O. violaceus had superior NPC and ion affinity compared with B. napus, and provided a novel, rapid, reliable method based on the plant’s electrophysiological information for real-time determination of the nutrient plunder capacity of plants.

First Report of Leaf Spot Disease Caused by Alternaria brassicae on Orychophragmus violaceus in China

Orychophragmus violaceus, belonging to the Brassicaceae family, is widely grown in many provinces of China as an ornamental plant and also as a green manure crop. In December 2019, field investigations showed that a leaf spot disease occurred on O.violaceus with 50% to 80% incidence in Huize City, Yunnan Province of China. Infected leaves showed symptoms of small black point spots in the early stage of onset. The lesions are distributed throughout the leaves and finaly expand to 10-15 mm in diameter after 10-15 days of onset. At this time, the lesions are gray to black, and some have round patterns, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. The leaves with typical lesion symptoms were sampled and photographed, and then subjected to isolate and characterize the pathogen. Six pure cultures (HEYA2; HEYA4; HEYC6; HEYD7; HEYD8; HEYD10) were obtained by single-hyphae isolation. On PCA medium, colony can reach 27 mm after 7d, at 25°C in darkness. Aerial hypha is cottony with white to pale gray color, while the colony reverse is fawn to dark. on V8 medium, conidiophore solitary or clustered, erect or knee-curved, occasionally branched, pare brown, separated, 82–130 × 5–9 µm. Conidia are solitary, straight or slightly curved, inverted rod-shaped, pare brown to brown, with 6-10 transverse septa, 0-5 oblique and longitudinal septa, columnar beak, conidial bodies (47.7-)69.3-103.8(-119.6)(11.2-)16.6-23.6(-27.8) µm. Beak septum, pare brown, (29.2-)34.4-72.4(-101.3)(4.2-)6.6-9.5(-11.3) µm. Morphologically these isolates resembled species belonging to genus Alternaria (Simmons, 2007). Genomic DNA of each culture was quickly extracted from mycelia using QS method (Chi et al. 2009). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2) and translation elongation factor -1α (TEF -1α) genes were amplified according described procedures (White et al. 1990; Berbee et al. 1999; Liu et al. 1999；Sung et al. 2007; Carbone & Kohn 1999). The sequences obtained in this study were deposited in GenBank with accession numbers: MW867245, MW867246, MW867247, MW867248, MW867249, MW867250, MW882913, MW882914, MW882915, MW882916, MW882917, MW882918, MW882919, MW882920, MW882921, MW882922, MW882923, MW882924, MW882925, MW882926, MW882927, MW882928, MW882929, MW882930. Phylogenetic analysis was conducted with combined sequences of the four loci, using the maximum likelihood method and the maximum parsimony method. In the phylogenetic tree, the six isolates and Alternaria brassicae (CBS 116528) clustered together with high bootstrap support values (MLBS=100; MPBS= 100). Based on both morphological characters and phylogenetic results, the isolates were identified as Alternaria brassicae. Pathogenicity test of isolate HEYA2 was carried out on the detached leaves in a dark thermostat incubator at 25°C. Five pots per leaf were inoculated with mycelia plugs (5 mm in diameter), another five pots were inoculated with pure agar plugs and used as the negative control. In addition, conidia suspension (105 conidia/ml) of isolate HEYD8 were sprayed on 3-month-old healthy plants grown in a greenhouse at 22 °C–28 °C. The plants sprayed with sterilized water were used as negative controls. The test was conducted three times. After 5-7 days, the leaves inoculated with other the conidia suspension or the mycelium plugs showed brown necrotic lesions that are similar to the symptoms observed in the field, but the controls remained healthy. The pathogen was reisolated and confirmed to be A. brassicae, completing Koch’s postulates. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on Orychophragmus violaceus in China.

Effects of Foliage Spraying with Sodium Bisulfite on the Photosynthesis of Orychophragmus violaceus

Sulphurous acid derived from sulfur dioxide (SO2) emission leads to the pollution of irrigation water and the inhibition of plant growth. The safe concentration threshold of NaHSO3 in plants should be clarified to promote agricultural production. In this study, Orychophragmus violaceus seedlings were used as experimental materials and five NaHSO3 concentrations (i.e., 0, 1, 2, 5, 10 mmol·L−1) were simultaneously sprayed on the leaf surface of different seedlings separately. Leaf physiology responses under different concentrations were analyzed. The NaHSO3 did not promote photosynthesis in O. violaceus under the 1 and 2 mmol·L−1 treatments. It was conducive to the net photosynthetic rate (PN), photorespiration rate (Rp), chlorophyll content, actual photochemical quantum yield (YII) and photochemical quenching (qP) under the 5 mmol·L−1 treatment. However, quantum yield of regulated energy dissipation (YNPQ) and nonphotochemical quenching (NPQ) were inhibited. Under the 10 mmol·L−1 treatment, PN, chlorophyll content, YII, qP, dark respiration rate (Rd) and electron transport rate (ETR) showed significant decreases, while the photorespiration portion (Sp) significantly increased. Our results demonstrated that NaHSO3 provided a sulfur source for plant growth and interfered with the redox reaction of the plant itself, and its role as a photorespiratory inhibitor might be masked.

Leaf spot disease of Orychophragmus violaceus caused by Alternaria tenuissima in China

Orychophragmus violaceus (L.) O. E. Schulz, also called February orchid or Chinese violet cress, belongs to the Brassicaceae family and is widely cultivated as a green manure and garden plant in China. During the prolonged rainy period in August 2020, leaf spot disease of O. violaceus was observed in the garden of Northeast Agricultural University, Harbin, Heilongjiang province. One week after the rainy days, the disease became more serious and the disease incidence ultimately reached approximately 80%. The disease symptoms began as small brown spots on the leaves, and gradually expanded to irregular or circular spots. As the disease progressed, spots became withered with grayish-white centers and surrounded by dark brown margins. Later on, the centers collapsed into holes. For severely affected plants, the spots coalesced into large necrotic areas and resulted in premature defoliation. No conidiophores or hyphae were present, and disease symptoms were not observed on other tissues of O. violaceus. To isolate the pathogen, ten leaves with typical symptoms were collected from different individual plants. Small square leaf pieces (5×5 mm) were excised from the junction of diseased and healthy tissues, disinfected in 75% ethanol solution for 1 min, rinsed in sterile distilled water, and then transferred to Petri dishes (9 cm in diameter) containing potato dextrose agar (PDA). After 3 days of incubation at 25 oC in darkness, newly grown-out mycelia were transferred onto fresh PDA and purified by single-spore isolation. Nine fungal isolates (NEAU-1 ~ NEAU-9) showing similar morphological characteristics were obtained and no other fungi were isolated. The isolation frequency from the leaves was almost 90%. On PDA plates, all colonies were grey-white with loose and cottony aerial hyphae, and then turned olive-green and eventually brown with grey-white margins. The fungus formed pale brown conidiophores with sparsely branched chains on potato carrot agar (PCA) plates after incubation at 25 oC in darkness for 7 days. Conidia were ellipsoidal or ovoid, light brown, and ranged from 18.4 to 59.1 × 9.2 to 22.3 µm in size, with zero to two longitudinal septa and one to five transverse septa and with a cylindrical light brown beak (n = 50). Based on the cultural and morphological characteristics, the fungus was tentatively identified as Alternaria tenuissima (Simmons 2007). Genomic DNA was extracted from the mycelia of five selected isolates (NEAU-1 ~ NEAU-5). The internal transcribed spacer region (ITS) was amplified and sequenced using primers ITS1/ITS4 (White et al., 1990). Blast analysis demonstrated that these five isolates had the same ITS sequence, and the ITS sequence of representative strain NEAU-5 (GenBank accession No. MW139354) showed 100% identity with the type strains of Alternaria alternata CBS916.96 and Alternaria tenuissima CBS918.96. Furthermore, the translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (GPD) of representative strain NEAU-5 were amplified and sequenced using primers EF1-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-5R (Sung et al., 2007), and Gpd1/Gpd2 (Berbee et al., 1999), respectively. The sequences of RPB2, GPD, and TEF of strain NEAU-5 were submitted to GenBank with accession numbers of MW401634, MW165223, and MW165221, respectively. Phylogenetic trees based on ITS, RPB2, GPD, and TEF were constructed with the neighbour-joining and maximum-likelihood algorithms using MEGA software version 7.0. The results demonstrated that strain NEAU-5 formed a robust clade with A. tenuissima CBS918.96 (supported by 99% and 96% bootstrap values) in the neighbour-joining and maximum-likelihood trees. As mentioned above, strain NEAU-5 produced seldomly branched conidial chains on PCA plates. The pattern is consistent with that of A. tenuissima (Kunze) Wiltshire, but distinct from that of A. alternata which could produce abundant secondary ramification (Simmons 2007). Thus, strain NEAU-5 was identified as A. tenuissima based on its morphology and phylogeny. Pathogenicity tests were carried out by inoculating five unwounded leaves with a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) on five different healthy plants cultivated in garden, and an equal number of leaves on the same plants inoculated with sterilized ddH2O served as negative controls. Inoculated and control leaves were covered with clear plastic bags for 3 days. After 6 days, small brown and irregular or circular spots were observed on all leaves inoculated with conidial suspension, while no such symptoms were observed in the control. The tests were repeated three times. Furthermore, the pathogenicity tests were also performed using 2-month-old potted plants in a growth chamber (28 oC, 90% relative humidity, 12 h/12 h light/dark) with two repetitions. Five healthy plants were inoculated by spraying 20 ml of a conidial suspension of strain NEAU-5 (approximately 106 conidia/ml) onto unwounded leaves. Five other healthy plants were inoculated with sterilized ddH2O as controls. After 7 days, similar symptoms were observed on leaves inoculated with strain NEAU-5, whereas no symptoms were observed in the control. The pathogen was reisolated from the inoculated leaves and identified as A. tenuissima by morphological and molecular methods. In all pathogenicity tests, A. tenuissima could successfully infect unwounded leaves of O. violaceus, indicating a direct interaction between leaves and A. tenuissima. It is known that high humidity and fairly high temperatures can favor the epidemics of Alternaria leaf spot (Yang et al., 2018), and this may explain why severe leaf spot disease of O. violaceus was observed after prolonged rain. Previously, it has been reported that Alternaria brassicicola and Alternaria japonica could cause leaf blight and spot disease on O. violaceus in Hebei and Jiangsu Provinces, China, respectively (Guo et al., 2019; Sein et al., 2020). Although these pathogens could lead to similar disease symptoms on the leaves of O. violaceus, it is easy to distinguish them by the morphological characteristics of conidiophores and ITS gene sequences. To our knowledge, this is the first report of A. tenuissima causing leaf spot disease of O. violaceus in China.