Anthracnose of Tribulus terrestris Caused by Colletotrichum truncatum in Northeast China.

Tribulus terrestris L. is an annual herbaceous medicinal plant of Zygophyllaceae, which is cultivated commercially in China. Subrotund or irregular gray, sunken, necrotic spots ranging from 2 to 9 mm were observed on diseased leaves of T. terrestris landrace in Fushun County, Liaoning Province of northeast China in July 2021, with more than 32% of the plants being infected in a 18-ha field. The symptoms first appeared on older leaves and gradually spread to younger leaves. The lesions developed a white center gradually and became perforated; multiple lesions could coalesce (Fig. 1). Ten symptomatic leaves were collected and the diseased tissues were cut into small pieces, immersed in 1% NaOCl for 2 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes at 25°C in darkness. Fifteen suspected Colletotrichum single-spore fungal isolates (JL1 to JL15) with consistent morphological characteristics were obtained, and isolate JL6 was selected for identification and pathogenicity testing. Colonies on PDA were flat with an entire margin, dense and white at first, then became dark gray with numerous black microsclerotia and formed a concentric circular pattern with aging. Conidia were single-celled, sickle-curved with a tapered tip and truncate base, ranging from 16.46 to 20.26 µm in length and 2.81 to 3.96 µm in width (n=100). Setae were dark brown, septate, straight with a slightly acute tip, 75.45 to 135.63×3.19 to 4.95 µm in size. Appressoria were dark brown, round or irregular, mostly in groups. All characteristics were consistent with the descriptions of C. truncatum (Damm et al. 2009). Further confirmation of the identification was determined according to methods described previously (Damm et al. 2009). The rDNA internal transcribed spacer region (OP364400, 585 bp), and actin (OP380867, 290 bp), beta-tubulin (OP380868, 498 bp), chitin synthase 1 (OP380869, 277 bp), glyceraldehyde-3-phosphate dehydrogenase (OP380870, 280 bp), and histone (OP380871, 411 bp) genes were amplified by PCR and sequenced (Carbone and Kohn 1999; Glass and& Donaldson 1995; Guerber et al. 2003; O'Donnell and Cigelnik 1997). BLAST results showed 98-100% similarity at 85-97% coverage compared to the corresponding sequences of the type strain CBS 151.35 (GU227862, GU227960, GU228156, GU228352, GU228254, and GU228058). Phylogenetic analysis combining all loci revealed that the isolate JL6 and the type strains of C. truncatum clustered in one group (Fig. 2). One-year-old healthy seedlings of T. terrestris (cultivar: landrace) were used for pathogenicity test. Suspension (1×105 conidia/mL) of isolate JL6 was sprayed on ten seedlings, and ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. All plants were kept at 28±1°C (12 h photoperiod), and were evaluated after 7 days. The inoculated plants showed lesions on the leaf surface, similar to those in the field, and the control remained symptomless. The pathogen was successfully reisolated and identified using the methods mentioned above. To our knowledge, this is the first report of C. truncatum causing anthracnose on T. terrestris, which will provide valuable information for designing strategies to manage anthracnose on T. terrestris.

Leaf Spot Caused by Boeremia linicola on Siberian ginseng in China.

Siberian ginseng (Eleutherococcus sessiliflorus (Rupr. & Maxim.) S. Y. Hu, Araliaceae), is a perennial medicinal plant that is widely cultivated in China. Leaf spot was observed in 2- and 3-year-old Siberian ginseng in Zuojia County (126°05'23.2″E, 44°03'09.5″N), northeast China, in August 2019. Polygonal or irregular black spots ranging from 2 to 9 mm in diameter were found on infected leaves, and each leaf had dozens of spots. The green color around the lesions gradually faded. As the disease progressed, the spots withered and multiple lesions merged into large disease spots, causing leaf wilting (Fig. 1). More than 38% of plants in one 25-ha field were infected in 2019. Fifteen diseased leaves were collected from those plants and cut into 5-mm pieces. The pieces were surface-disinfected by immersion in 1% NaOCl for 2 min and then rinsing twice with sterile distilled water. The leaf pieces were placed on acidified potato dextrose agar (PDA, pH 4.7) in Petri plates, and incubated in the dark at 25°C. Nineteen isolates were obtained and all were purified from a single spore in water agar. Isolate CWJ7 was randomly selected for identification and pathogenicity testing. The colonies on PDA were olivaceous gray to olivaceous black, velvet, with dense hyphae and a scalloped or irregular margin. The reverse side was gray-black and surrounded by tawny halos. The conidia were aseptate and variable in shape and dimension: piriform, columnar, drop-shaped, dumbbell-shaped or oval, measuring 4.90 (7.03) 9.50 × 2.10 (2.78) 3.40 µm (n=100), and chlamydospores were absent. Black pycnidia (132.2-241.5 µm in diameter) appeared after 7 days. The pathogen was initially identified as Phoma or Phoma-like (Boerema et al. 2004). Further confirmation was also determined by sequencing the nuclear ribosomal internal transcribed spacer region (GenBank accession no. MT912950), 28S ribosomal RNA gene (MT912968), and genes encoding ß-tubulin (MT920618), the second largest subunit of RNA polymerase II (MT920619) and translation elongation factor (MT946526) (de Hoog and Gerrits van den Ende 1998; Rehner & Samuels 1994; Liu et al. 1999; Vilgalys & Hester 1990), and Blast searches showed 90%-100% homology with GU237754, GU237938, KT389780, KT389575, and KY484705, respectively. In a phylogenetic analysis combining all loci, CWJ7 and the type strains of Boeremia linicola clustered in one group (Fig. 2). Based on its morphological characteristics and phylogenetic analysis, isolate CWJ7 was identified as B. linicola as revised in 2019 (Jayawardena et al. 2019). Healthy 2-year-old plants were used for pathogenicity testing. The leaves of nine potted plants (one plant per pot, three plants per replicate) were spray-inoculated with a suspension of conidia (1×105 spores/ml) from colonies on PDA for 7 days and cultured for 48 h under continuous black light. Nine plants were sprayed with sterile water as the control. This experiment was repeated twice. All plants were cultured in a greenhouse (25°C, 12-h photoperiod, 78% relative humidity). Clear plastic bags were used to maintain high humidity. After 7 days, the inoculated plants showed lesions on the leaves, similar to those observed in the field. The control plants remained symptomless. The pathogen was reisolated and identified by sequencing. This is the first report of B.linicola causing Siberian ginseng leaf spot, and a new record of this species in China. This disease poses a threat to production and management strategies should be developed.

Occurrence of Dactylonectria torresensis Causing Root Rot on Astragalus membranaceus in northeastern China.

Astragalus membranaceus Bunge (Fabaceae) is a perennial medicinal herb widely cultivated in China. In June 2018, root rot was observed on two-year-old A. membranaceus plants in Chaoyangshan town (northeastern China). In a 40-ha field, over 40% of the plants exhibited root rot and the infected area ranged from 10 to 70% of the roots. The roots first exhibited circular or irregular brown, sunken and necrotic lesions, and finally multiple lesions coalesced. The infected root surface was destroyed, showing rusty and dry rot (Fig. 1). Symptoms were concentrated in the main roots (Carlucci et al. 2017). The aboveground parts of infected plants did not initially show symptoms but gradually wilted; 7.6% of the plants died when root decay became severe. Infected roots were not used for processing and were not marketable. Ten infected roots were collected from May to October 2018 from the above location. The diseased root tissue was cut into 25 mm3 pieces, immersed in 1% NaOCl for 2 minutes, rinsed three times with sterile water and placed on water agar in Petri plates. After 15 days of incubation at 20°C, 11 single-spore isolates were obtained. Isolates HQ1 and HQ2 were randomly selected for morphological and molecular identification. Colonies grown for 10 days produced yellow, cottony to felty aerial mycelium on potato dextrose agar. Conidiophores originating laterally or terminally from the mycelium were solitary to loosely aggregated and unbranched or sparsely branched. Macroconidia predominated and were cylindrical, with a tendency to gradually widen towards the tip; 1- to 3-septate; and 20.2 to 31.0 × 3.0 to 6.7 µm (n=100). Microconidia had mostly 0¬- to 1-septate and 8.6 to 16.7 × 1.9 to 5.1 µm (n=100) (Fig. 1). Chlamydospores were rare, but occasional chlamydospore chains were observed. The isolates were tentatively identified as Dactylonectria torresensis (Cabral et al. 2012a). Further confirmation of the two isolates was conducted by DNA sequencing of the internal transcribed spacer (ITS, GenBank accession no. MN558983 and MN558984), ß-tubulin (TUB, MN561692 and MN561693), histone 3 (HIS3, MN561694 and MN561695), and translation elongation factor (TEF, MN561696 and MN561697) genes (Cabral et al. 2012b). These sequences had 99 to 100% match with D. torresensis (JF735362 for ITS, JF735492 for TUB, JF735681 for HIS3 and JF735870 for TEF). Phylogenetic trees based on analyses of a concatenated alignment of all loci grouped these isolates into the D. torresensis clade (Fig. 2). The same two isolates were tested for pathogenicity. Healthy two-year-old plants were taken from the field, and their roots were disinfected with 75% alcohol for 3 minutes, rinsed with sterile water three times, immersed in a 1×105/ml spore suspension or sterile water (control) for 10 minutes, transferred to a tray filled with sterile sand and placed in a greenhouse (12 h photoperiod, 25°C). Twelve plants grown in three pots were used for each isolate, and the same number of plants were inoculated as a control. This experiment was repeated three times. After one month, inoculated plant roots showed the same symptoms as those observed in the field, while the controls remained symptomless and no pathogen was recovered. The same fungus was reisolated from all the infected plants and confirmed by sequencing all of the above genes. This is the first report of D. torresensis causing root rot in A. membranaceus in China. The occurrence of this disease poses a threat, and management strategies need to be developed.

[Photosynthetic characteristics and active ingredients differences of Asarum heterotropoides var. mandshuricum under different light irradiance].

Chlorophyll content,leaf mass to per area,net photosynthetic rate and bioactive ingredients of Asarum heterotropoides var. mandshuricum,a skiophyte grown in four levels of solar irradiance were measured and analyzed in order to investigate the response of photosynthetic capability to light irradiance and other environmental factors. It suggested that the leaf mass to per area of plant was greatest value of four kinds of light irradiance and decreasing intensity of solar irradiance resulted in the decrease of leaf mass to per area at every phenological stage. At expanding leaf stage,the rate of Chla and Chlb was 3. 11 when A. heterotropoides var. mandshuricum grew in full light irradiance which is similar to the rate of heliophytes,however,the rate of Chla and Chlb was below to 3. 0 when they grew in shading environment. The content of Chla,Chlb and Chl( a+b) was the greatest value of four kinds of light irradiance and decreasing intensity of solar irradiance resulted in its decreasing remarkably( P<0. 05). The rate of Chla and Chlb decreased but the content of Chla,Chlb and Chl( a+b) increased gradually with continued shading. The maximum value of photosynthetically active radiation appeared at 10: 00-12: 00 am in a day. The maximum value of net photosynthetic rate appeared at 8: 30-9: 00 am and the minimum value appeared at 14: 00-14: 30 pm at each phenological stage if plants grew in full sunlight. However,when plants grew in shading,the maximum value of net photosynthetic rate appeared at about 10: 30 am and the minimum value appeared at 12: 20-12: 50 pm at each phenological stage. At expanding leaf stage and flowering stage,the average of net photosynthetic rate of leaves in full sunlight was remarkably higher than those in shading and it decreased greatly with decreasing of irradiance gradually( P < 0. 05). However,at fruiting stage,the average of net photosynthetic rate of leaves in full sunlight was lower than those in 50% and 28% full sunlight but higher than those in 12% full sunlight. All photosynthetic diurnal variation parameters of plants measured in four kinds of different irradiance at three stages were used in correlation analysis. The results suggested that no significant correlation was observed between net photosynthetic rate and photosynthetically active radiation,and significant negative correlation was observed between net photosynthetic rate and environmental temperature as well as vapor pressure deficit expect for 12% full sunlight. Positive correlation was observed between net photosynthestic rate and relative humidity expect for 12% full sunlight. Significant positive correlation was observed between net photosynthetic rate and stomatal conductance in the four light treatments. Only,in 12% full sunlight,the net photosynthetic rate was significantly related to photosynthetically active radiation rather than related to environmental temperature,vapor pressure deficit and relative humidity. In each light treatment,a significant positive correlation was observed between environmental temperature and vapor pressure deficit,relative humidity as well as stomatal conductance. Volatile oil content was 1. 46%,2. 16%,1. 56%,1. 30% respectively. ethanol extracts was 23. 44%,22. 45%,22. 18%,21. 12% respectively. Asarinin content was 0. 281%,0. 291%,0. 279% and 0. 252% respectively. The characteristic components of Asarum volatile oil of plant in different light treatments did not change significantly among different groups.

Response and tolerance of root border cells to aluminum toxicity in soybean seedlings.

Root border cells (RBCs) and their secreted mucilage are suggested to participate in the resistance against toxic metal cations, including aluminum (Al), in the rhizosphere. However, the mechanisms by which the individual cell populations respond to Al and their role in Al resistance still remain unclear. In this research, the response and tolerance of RBCs to Al toxicity were investigated in the root tips of two soybean cultivars [Zhechun No. 2 (Al-tolerant cultivar) and Huachun No. 18 (Al-sensitive cultivar)]. Al inhibited root elongation and increased pectin methylesterase (PME) activity in the root tip. Removal of RBCs from the root tips resulted in a more severe inhibition of root elongation, especially in Huachun No. 18. Increasing Al levels and treatment time decreased the relative percent viability of RBCs in situ and in vitro in both soybean cultivars. Al application significantly increased mucilage layer thickness around the detached RBCs of both cultivars. Additionally, a significantly higher relative percent cell viability of attached and detached RBCs and thicker mucilage layers were observed in Zhechun No. 2. The higher viability of attached and detached RBCs, as well as the thickening of the mucilage layer in separated RBCs, suggest that RBCs play an important role in protecting root apices from Al toxicity.

[The expression of substance P in airway mucosa of guinea pigs with repetitive esophageal stimulation by hydrochloric acid].

OBJECTIVE: To observe the expression of substance P (SP) in the airway mucosa of guinea pigs with repetitive esophageal stimulation by hydrochloric acid (HCL). METHODS: Twenty adult guinea pigs were randomly divided into 2 groups (n = 10 each): (1) The HCL model group: On the day of experimentation, guinea pigs were maintained under ketamine anesthesia. A 5F catheter was inserted orally into the lumen of the middle and lower esophagus. The esophagus of each animal was perfused with HCl-P for 20 min/d for 14 d. (2) The PBS control group: The esophagus of each animal was perfused with PBS instead. The bronchial responsiveness to Ach given intravenously with increasing doses (3.125, 6.25, 12.5, 25, 50, 100 microg/kg) was measured after the last perfusion. The left lung was isolated for pathological examination. Lung sections were stained with hematoxylin and eosin, and other sections were prepared for immunohistochemistry using monoclonal antibodies against SP. RESULTS: In response to increasing doses of ACh, all guinea pigs showed dose-dependent increases in R(L). However, when the dose of ACh was increased to 25 microg/kg, the airway responsiveness increased significantly in the HCl-P model animals compared with the PBS control group (t values = 43.057, 51.410, 57.359 respectively, all P<0.01). The mean gray values of SP decreased significantly in the tracheal epithelia and the distal airway walls of the model group compared with the PBS control group (t values = 3.44, 2.16 respectively, all P<0.01). CONCLUSION: There was airway neurogenic inflammation in guinea pigs with repetitive esophageal stimulation by HCL, which maybe closely related to the pathogenesis of gastro-esophageal reflux disease.