First Report of Paraphoma chrysanthemicola Causing Crown and Leaf Rot of Atractylodes lancea in China

Atractylodes lancea is an important traditional Chinese medicinal plant whose rhizome is used for treating complaints such as rheumatic diseases, digestive disorders, night blindness and influenza. Jiangsu Province is the optimal cultivation location for high-quality A. lancea rhizome. Since June 2019, symptoms of crown rot and leaf rot were observed in about 10-20% of the A. lancea in a plantation (31° 36' 1" N, 119° 6' 40" W) in Lishui, Jiangsu, China. Lesions occurred on the stem near the soil line and on the leaves (Fig. 1A). Disease incidence reached approximately 80-90% by September, 2021 (Fig. 1B) and resulted in severe loss of rhizome and seed yields. For pathogen isolation, ten samples of symptomatic stem segments and ten diseased leaves were collected, surface-sterilized using 5% NaClO solution, rinsed with sterile water, cut into 0.5-2 cm segments, and plated to potato dextrose agar (PDA), and then incubated at 30°C in darkness. Pure cultures of four isolates showing morphological characteristics of Paraphoma spp. were obtained, identified as a single P. chrysanthemicola strain, and named LSL3f2. Newly formed colonies initially consisted of white mycelia; the five-day-old colonies developed a layer of whitish grey mycelia with a grey underside. 20-day-old colonies had white mycelium along the margin and with a faint yellow inner circular part with irregular radial furrows, and the reverse side looking caramel and russet (Fig. 1C). Pycnidia were subglobose (diameter: 5 to 15 μm; Fig. 1D). Unicellular, bicellular or strings of globose or subglobose chlamydospores developed from hyphal cells (Fig. 1E and 1F). The internal transcribed spacer (ITS) region and large subulin-28S of LSL3f2 were cloned using primers ITS1/ITS4 and LR0R/LR7 (Aveskamp et al. 2010, Li et al. 2013), and deposited in GenBank (OK559658 and OK598973, respectively). BLASTn search and phylogenetic analysis showed the highest identity between LSL3f2 and P. chrysanthemicola sequences (Fig. 1G) and confirmed LSL3f2 as P. chrysanthemicola. Koch’s postulates were completed using one-month-old vegetatively propagated A. lancea plantlets growing on autoclaved vermiculite/peat mixture at 26°C with a light/dark cycle of 12/12 hours. Each plantlet was inoculated with 5 ml of conidial suspension in water (1 × 108 cfu/ml) by applying to soil close to the plantlet, with sterile water used as a mock control (n = 10). By 20 days post-inoculation, inoculated plantlets showed a range of disease symptoms consistent to those observed in infested fields (Fig. 1H). Pathogenicity was additionally confirmed using detached leaves inoculated with a colonized agar plug of LSL3f2 or an uninoculated control comparison (diameter = 5 mm) and incubated at 26℃ in the dark. Five to seven days post-inoculation, detached leaves showed leaf rot symptoms including lesions, yellowing and withering consistent with those in infested fields, while control leaves remained healthy (n = 10, Fig. 1I). The pathogen was reisolated from the diseased plantlets and detached leaves, in both cases demonstrating the micromorphological characteristics of LSL3f2. P. chrysanthemicola has been reported to cause leaf and crown rot on other plants such as Tanacetum cinerariifolium (Moslemi et al. 2018), and leaf spot on A. japonicain (Ge et al. 2016). However, this is the first report of P. chrysanthemicola causing crown and leaf rot on A. lancea in China.

Phytonutrients and Metabolism Changes in Topped Radish Root and Its Detached Leaves during 1 °C Cold Postharvest Storage

Glucosinolates, lipid-soluble vitamins E and K contents, primary metabolites and plant hormones were analyzed from topped radish root and detached leaf during storage at 1 °C. The topped root was analyzed at 0, 5, 15, 30, and 90 days after storage while the detached leaf was analyzed at 0, 5, 15, 30, and 45 days in an airtight storage atmosphere environment. The results showed that aliphatic glucosinolates were gradually decreased in leaf but not in root. There was a highly significant correlation between tryptophan and 4-methoxyindoleglucobrassicin in both tissues (r = 0.922, n = 10). There was no significant difference in vitamins E and K in leaf and root during storage. Plant hormones partially explained the significantly changed metabolites by tissue and time, which were identified during cold storage. Phenylalanine, lysine, tryptophan, and myo-inositol were the most important biomarkers that explained the difference in leaf and root tissue during cold storage. The most different metabolism between leaf and root tissue was starch and sucrose metabolism. Therefore, different postharvest technology or regimes should be applied to these tissues.

Alternaria alternata as a Seed-Transmitted Pathogen of Sida hermaphrodita (Malvaceae) and Its Suppression by Aureobasidium pullulans

Background: Sida hermaphrodita (Virginia fanpetals) was introduced to Poland nearly 70 years ago as a potential fodder plant, and it is gaining importance as an energy crop. Alternaria alternata transmitted by seeds may exert a negative effect on the health of Virginia fanpetals plants. Methods: The virulence of the A. alternata pathogen, isolated from Virginia fanpetals seeds, was tested on detached leaves of Virginia fanpetals plants. The isolates were identified as A. alternata based on partial sequence analysis of Alta1, TEF1a and gdp genes and the ITS 1–5.8SrDNA–ITS 2 region. Pathogen transmission from seeds to seedlings and the influence of seed dressing with a suspension of Aureobasidium pullulans on seedling health were analyzed in a greenhouse experiment. Results: Three of the nine analyzed A. alternata isolates were highly pathogenic for S. hermaphrodita. The initial symptoms of leaf infection were small, round dark brown or black spots which grew into larger dark brown spots surrounded by a chlorotic halo. Alternaria alternata was re-isolated from inoculated plants and was identified as the causal agent of Alternaria leaf spot disease. In the greenhouse experiment, S. hermaphrodita seeds dressed with a suspension of A. pullulans and inoculated with A. alternata produced a higher number of seedlings with a higher health status than non-dressed seeds. Conclusions: The study demonstrated that A. alternata is transmitted from infected S. hermaphrodita seeds to developing plants and biological control limits this phenomenon.

Identification and pathogenicity of Alternaria species associated with leaf blotch disease and premature defoliation in French apple orchards

Leaf blotch caused by Alternaria spp. is a common disease in apple-producing regions. The disease is usually associated with one phylogenetic species and one species complex, Alternaria alternata and the Alternaria arborescens species complex (A. arborescens SC), respectively. Both taxa may include the Alternaria apple pathotype, a quarantine or regulated pathogen in several countries. The apple pathotype is characterized by the production of a host-selective toxin (HST) which is involved in pathogenicity towards the apple. A cluster of genes located on conditionally dispensable chromosomes (CDCs) is involved in the production of this HST (namely AMT in the case of the apple pathotype). Since 2016, leaf blotch and premature tree defoliation attributed to Alternaria spp. have been observed in apple-producing regions of central and south-eastern France. Our study aimed to identify the Alternaria species involved in apple tree defoliation and assess the presence of the apple pathotype in French orchards. From 2016 to 2018, 166 isolates were collected and identified by multi-locus sequence typing (MLST). This analysis revealed that all these French isolates belonged to either the A. arborescens SC or A. alternata. Specific PCR detection targeting three genes located on the CDC did not indicate the presence of the apple pathotype in France. Pathogenicity was assessed under laboratory conditions on detached leaves of Golden Delicious and Gala apple cultivars for a representative subset of 28 Alternaria isolates. All the tested isolates were pathogenic on detached leaves of cultivars Golden Delicious and Gala, but no differences were observed between the pathogenicity levels of A. arborescens SC and A. alternata. However, the results of our pathogenicity test suggest that cultivar Golden Delicious is more susceptible than Gala to Alternaria leaf blotch. Implications in the detection of the Alternaria apple pathotype and the taxonomic assignment of Alternaria isolates involved in Alternaria leaf blotch are discussed.

Zinc uptake and distribution in ivy (Hedera helix L.) leaves

Detached leaves of ivy (Hedera helix L.) were used as a model for the study of zinc uptake and transport in vascular plants. By the uptake via the surface of fully immersed leaves in 25 % Hoagland nutrient media (HM) spiked with 65ZnCl2 (50 μmol/dm3 ZnCl2), concentration in leaves 4.98 μg Zn/g (dry wt.), i. e. 2.6 μg Zn/dm2 leaf area after 7d exposition were obtained. By the uptake via immersed stalks of not immersed (transpiring) leaves concentrations up to 370 μg Zn/g (dry wt.) were obtained. When Zn enters into detached leaves via the surface of immersed leaf blades, zinc is uniformly distributed in leaf blades and leaf stalks. When zinc enters detached leaves via immersed stalks of non-immersed transpiring leaves, only small part of zinc is transported to leaf blades and the prevailing part remains in leaf stalks. Stalks act as a trap, able to prevent other leaf tissues against inhibitory effects of high Zn concentrations. Mineral nutrient salts in solutions mobilize Zn trapped in leaf stalks and facilitate Zn transport by transpiration stream to leaf blades, what means that Zn in stalks is bound in ion-exchageable forms.

Metabolic Profile and Mycoherbicidal Activity of Three Alternaria alternata Isolates for the Control of Convolvulus arvensis, Sonchus oleraceus, and Xanthium strumarium

Alternaria alternata isolates C1, S1, and X3 were isolated respectively from the weeds Convolvulus arvensis, Sonchus oleraceus, and Xanthium strumarium in Algiers during 2016 and identified by morphological and molecular analyses. The aim of this investigation was to chemically characterize the exometabolome of these fungi and to evaluate the myco-herbicidal potential of their culture filtrates, crude extracts, or fractions towards target weeds. Results revealed a great heterogeneity in the biochemical profiles of the exometabolome with the remarkable presence of two compounds: tenuazonic acid (TeA) and triprenyl phenol-7 (SMTP-7). To the best of our knowledge, SMTP-7—found in all the isolates—as well as 12-methoxycitromycin detected in the culture filtrate of isolate C1, have never been reported to be produced by A. alternata. Some fractions of isolates C1 and S1 showed symptoms (necrosis and chlorosis) on the detached leaves of C. arvensis and S. oleraceus, respectively with up to 100% phytotoxic effect at low concentration. In conclusion, biochemical characterization revealed great difference of C1, S1, and X3 exometabolomethat is likely to explain the difference in their phytotoxic activity. Some fractions (d1, e1, h1, i1, a2, and f2) of isolates C1 and S1 of A. alternata caused severe necrosis and chlorosis on the injured detached leaves of C. arvensis and S. oleraceus, respectively.

Screening of indigenous rhizobacteria as potential biological control against faba bean (Vicia faba L.) gall disease caused by Olpidium viciae

Background Emerged faba bean gall disease attacks the stem and leaves of the plant and results in complete crop losses. This study was initiated to screen multi-trait rhizobacteria for their antagonistic efficacy under in-vitro and in-vivo conditions against Olpidium viciae to control the effect of gall disease on faba bean. Sixty antagonistic isolates were first examined for their morphological, biochemical, and phenotypic traits. Results Pseudomonas fluorescens AAUPF62, P. aeruginosa AAUS31, Bacillus AAUMF42, and Bacillus AAUAm28 showed greater than 68, 62, 57, and 54% suppression of O. viciae in dual culture, volatile metabolites, culture filtrate assay, and detached leaves experiments, respectively. The in-vivo study revealed that early treatment of the crop with P. aeruginosa AAUS31 reduced severity by 63% (in FB-Obse) and 54% (in FB-26869) faba bean varieties. The co-inoculation of P. fluorescens AAUPF62 and P. aeruginosa AAUS31 significantly enhanced the shoot (P = 0.003; mean = 122 cm) and root (P = 0.018; mean = 94 cm) length, increased shoot dry weight by 8 factors (83 g pot −1), and reduced final disease severity by 92% in FB-Obse variety. Conclusions The results revealed that P. fluorescens AAUPF62 and P. aeruginosa AAUS31 strains could be the potential antagonistic agents of gall disease. The use and early treatment of moderately resistant faba bean varieties by co-inoculation of synergistic potential bioagents were recommended.

How many begomovirus copies are acquired and inoculated by its vector, whitefly (Bemisia tabaci) during feeding?

Begomoviruses are transmitted by whitefly (Bemisia tabaci Gennadius, Hemiptera: Aleyrodidae) in a persistent-circulative way. Once B. tabaci becomes viruliferous, it remains so throughout its life span. Not much is known about the copies of begomoviruses ingested and/or released by B. tabaci during the process of feeding. The present study reports the absolute quantification of two different begomoviruses viz. tomato leaf curl New Delhi virus (ToLCNDV, bipartite) and chilli leaf curl virus (ChiLCV, monopartite) at different exposure of active acquisition and inoculation feeding using a detached leaf assay. A million copies of both the begomoviruses were acquired by a single B. tabaci with only 5 min of active feeding and virus copy number increased in a logarithmic model with feeding exposure. Whereas, a single B. tabaci could inoculate 8.21E+09 and 4.19E+11 copies of ToLCNDV and ChiLCV, respectively in detached leaves by 5 min of active feeding. Virus copies in inoculated leaves increased with an increase in feeding duration. Comparative dynamics of these two begomoviruses indicated that B. tabaci adult acquired around 14-fold higher copies of ChiLCV than ToLCNDV 24 hrs post feeding. Whereas, the rate of inoculation of ToLCNDV by individual B. tabaci was significantly higher than ChiLCV. The study provides a better understanding of begomovirus acquisition and inoculation dynamics by individual B. tabaci and would facilitate research on virus-vector epidemiology and screening host resistance.

Metalaxyl Resistance of Phytophthora palmivora Causing Durian Diseases in Thailand

Thailand is the leading producer and exporter of durians worldwide. Serious diseases in durians include root rot, stem rot, and fruit rot, which are caused by Phytophthora palmivora, P. nicotianae, and Pythium cucurbitacearum, respectively. Thai farmers have applied fungicides for more than 20 years to control rot, but it remains difficult to control. Thus, the monitoring of fungicide-resistance development in pathogens is important for disease management. Pathogens were isolated from naturally infected durians between 2016 and 2017 in southern Thailand. The sequences of the internal transcribed spacer (ITS) and 5.8S regions of rDNA were used for the identification of their species. Seventeen out of twenty isolates were confirmed to be P. palmivora. All the isolates were tested for mycelium-growth sensitivity to metalaxyl, azoxystrobin, and dimethomorph. The results showed that nine isolates were resistant to metalaxyl with the 50% effective concentration (EC50) higher than 100 mg L−1. By contrast, all the isolates were sensitive to both azoxystrobin and dimethomorph, with EC50 < 1 mg L−1. Metalaxyl-resistant isolates were not controlled (−25.6% to 22.2%) by the treatment of the detached leaves of ‘Monthong’ durian with 100 mg L−1 metalaxyl prior to inoculation, but all the metalaxyl-sensitive and moderately metalaxyl-resistant isolates were better controlled (33.0% to 62.6%). These results clearly indicate that metalaxyl-resistant strains are present in the populations of P. palmivora in Thailand.

NOXA Is Important for Verticillium dahliae’s Penetration Ability and Virulence

NADPH oxidase (Nox) genes are responsible for Reactive Oxygen Species (ROS) production in living organisms such as plants, animals, and fungi, where ROS exert different functions. ROS are critical for sexual development and cellular differentiation in fungi. In previous publications, two genes encoding thioredoxin and NADH-ubiquinone oxidoreductase involved in maintaining ROS balance were shown to be remarkably induced in a highly versus a weakly aggressive Verticillium dahliae isolate. This suggested a role of these genes in the virulence of this pathogen. NoxA (NADPH oxidase A) was identified in the V. dahliae genome. We compared in vitro expression of NoxA in highly and weakly aggressive isolates of V. dahliae after elicitation with extracts from different potato tissues. NoxA expression was induced more in the weakly than highly aggressive isolate in response to leaf and stem extracts. After inoculation of potato detached leaves with these two V. dahliae isolates, NoxA was drastically up-regulated in the highly versus the weakly aggressive isolate. We generated single gene disruption mutants for NoxA genes. noxa mutants had significantly reduced virulence, indicating important roles in V. dahliae pathogenesis on the potato. This is consistent with a significant reduction of cellophane penetration ability of the mutants compared to the wild type. However, the cell wall integrity was not impaired in the noxa mutants when compared with the wild type. The resistance of noxa mutants to oxidative stress were also similar to the wild type. Complementation of noxa mutants with a full length NoxA clones restored penetration and pathogenic ability of the fungus. Our data showed that NoxA is essential for both penetration peg formation and virulence in V. dahliae.