A new Panax notoginseng Leaf Spot Caused by Boeremia linicola in Yunnan, China.

Panax notoginseng-also known as Tianqi and Sanqi-is one of the most highly valued medicinal perennial herbs in the world (Wang et al. 2016). In August 2021, leaf spot was observed on P. notoginseng leaves in Lincang sanqi base (23º43´10˝N, 100º7´32˝E, 13.33 hm2). Symptoms expanded from water soaked areas on the leaves to form irregular round or oval leaf spots with transparent or grayish-brown centers containing black granular matter, with an incidence of 10 to 20%. To identify the causal agent, ten symptomatic leaves were randomly selected from ten P. notoginseng plants. Symptomatic leaves were cut into small pieces (5 mm2) with asymptomatic tissue margins, disinfected in 75% ethanol for 30s and in 2% sodium hypochlorite for 3 min, and rinsed three times with sterile distilled water. The tissue portions were placed on potato dextrose agar (PDA) plates incubated at 20℃ with a 12 h light/dark photoperiod. Seven pure isolates were obtained with similar colony morphology, dark gray (top view) or taupe (back view) coloration, with flat and villous surfaces. Pycnidia were globose to subglobose, glabrous or with few mycelial outgrowths, dark brown to black, 22.46 to 155.94 (av. 69.57) µm × 18.20 to 130.5 (av. 57.65) µm (n=50) in size. Conidia were ellipsoidal to cylindrical, thinwalled, smooth, hyaline, aseptate, and measured 1.47 to 6.81 (av. 4.29) µm long and 1.01 to 2.97 (av. 1.98) µm thick (n=100). The isolated strains were preliminarily identified as Boeremia sp. based on the morphological characteristics of colonies and conidia. (Aveskamp et al. 2010; Schaffrath et al. 2021). To confirm pathogen identity, the total genomic DNA of two isolates (LYB-2 and LYB-3) was extracted using the T5 Direct PCR kit. The internal transcribed spacer (ITS), 28S large subunit nrRNA gene (LSU), and ß-tubulin (TUB2) gene regions were PCR-amplified using primers ITS1/ITS4, LR0Rf/LR5r, and BT2F/BT4R (Chen et al. 2015), respectively. Sequences have been deposited in GenBank (ON908942-ON908943 for ITS, ON908944-ON908945 for LSU, ON929285-ON929286 for TUB2). BLASTn searches of generated DNA sequences from 2 purified isolates (LYB-2 and LYB-3) against GenBank showed high similarity (>99%) with the sequences of Boeremia linicola. Moreover, a phylogenetic tree was constructed based on the neighbor-joining method in MEGA-X (Kumar et al. 2018) and revealed that the 2 isolates were closest to B. linicola (CBS 116.76). Pathogenicity tests were conducted with the 2 isolates (LYB-2 and LYB-3) as described by Cai et al. (2009) with slight modifications. Each isolate was inoculated with three healthy annual P. notoginseng plants, and each leaf was inoculated with three drops of conidia suspension (106 spores/mL). Three P. notoginseng plants inoculated with sterile water were used as controls. All plants were covered with plastic bags incubated in a greenhouse (20℃, 90%RH, 12 h light/dark photoperiod). Fifteen days post-inoculation, all inoculated leaves showed similar lesions, and the symptoms were identical to those in the field. The pathogen was reisolated from symptomatic leaf spots, and the colony characteristics were identical to the original isolates. Control plants remained healthy, and no fungus was re-isolated. Morphological characteristics, sequence alignment and pathogenicity tests confirmed that B. linicola was the cause of P. notoginseng leaf spot disease. This is the first report of B. linicola causing leaf spot on P. notoginseng in Yunnan, China. The identification of B. linicola as the causal agent of the observed leaf spot on P. notoginseng is critical to the prevention and control of this disease in the future.

Autotoxic Ginsenoside Stress Induces Changes in Root Exudates to Recruit the Beneficial Burkholderia Strain B36 as Revealed by Transcriptomic and Metabolomic Approaches.

Plants can recruit beneficial microbes to help improve their fitness under abiotic or biotic stress. Our previous studies found that Panax notoginseng could enrich beneficial Burkholderia sp. B36 in the rhizosphere soil under autotoxic ginsenoside stress. Here, we clarified that ginsenoside stress activated the phenylpropanoid biosynthesis and α-linolenic acid metabolism pathways of roots to increase the secretion of cinnamic acid, 2-dodecenoic acid, and 12-oxo-phytodienoic acid. These metabolites could promote the growth of B36. Importantly, cinnamic acid could simultaneously promote the chemotaxis and growth of B36, enhance the colonization of B36 in the rhizosphere, and eventually increase the survival rate of P. notoginseng. Overall, the plants could promote the growth and colonization of beneficial bacteria through key metabolites in root exudates under autotoxin stress. This finding will facilitate the practical application of beneficial bacteria in agricultural production and lead to successful and reproducible biocontrol efficacy by the exogenous addition of key metabolites.

Effects of different soil moisture on the growth, quality, and root rot disease of organic Panax notoginseng cultivated under pine forests.

The under-forest economy in the agroforestry system can improve land use efficiency, protect ecological environment, and promote arable land sustainable development. However, the effects of soil moisture in the forest and irrigation strategies on the healthy growth of intercropping crops are still incomplete. Here, considering the organic Panax notoginseng cultivated under pine forests (PPF) as the research object, we explored the effects of different soil moisture on the physiological state, yield, quality and disease occurrence of PPF. Our results suggested that 80-85% and 95-100% field capacity (FC) treatments were more conducive to increased photosynthetic rate and biomass accumulation of PPF, but 50-55% and 65-70% FC treatments were more conducive to the accumulation of saponins in PPF leaves. Notably, the root rot index of PPF was highest under 95-100% FC (19.51) treatment, significantly higher than that under 65-70% FC (8.44) and 80-85% FC (10.21) treatments. Further, the rhizosphere microorganisms of PPF under different soil moisture treatments were sequenced, and the sequencing data analysis revealed that high soil moisture (95-100% FC) could destroy the microbial diversity balance and cause the accumulation of pathogens (Fusarium oxysporum and Ilyonectria radicicola), leading to a high incidence of root rot. The incidence of PPF root rot was negatively correlated with rhizosphere microbial diversity. Overall, our results highlight that the quantitative irrigation (80-85% FC) is conducive to maintaining the balance between yield, saponin content and disease occurrence of PPF, providing a practical basis for PPF irrigation strategy and promoting the sustainable development of PPF agroforestry system.

Foliar Pathogen Infection Manipulates Soil Health through Root Exudate-Modified Rhizosphere Microbiome.

Negative plant-soil feedback (NPSF) due to the buildup of soilborne pathogens in soil is a major obstacle in sustainable agricultural systems. Beneficial rhizosphere microfloras are recruited by plants, and mediating this has become a strategic priority to manipulate plant health. Here, we found that foliar infection of Panax notoginseng by Alternaria panax changed plant-soil feedback from negative to positive. Foliar infection modified the rhizosphere soil microbial community and reversed the direction of the buildup of the soilborne pathogen Ilyonectria destructans and beneficial microbes, including Trichoderma, Bacillus, and Streptomyces, in rhizosphere soil. These beneficial microbes not only showed antagonistic ability against the pathogen I. destructans but also enhanced the resistance of plants to A. panax. Foliar infection enhanced the exudation of short- and long-chain organic acids, sugars, and amino acids from roots. In vitro and in vivo experiments validated that short- and long-chain organic acids and sugars play dual roles in simultaneously suppressing pathogens but enriching beneficial microbes. In summary, foliar infection could change root secretion to drive shifts in the rhizosphere microbial community to enhance soil health, providing a new strategy to alleviate belowground disease in plants through aboveground inducement. IMPORTANCE Belowground soilborne disease is the main factor limiting sustainable agricultural production and is difficult to manage due to the complexity of the soil environment. Here, we found that aboveground parts of plants infected by foliar pathogens could enhance the secretion of organic acids, sugars, and amino acids in root exudates to suppress soilborne pathogens and enrich beneficial microbes, eventually changing the plant and soil feedback from negative to positive and alleviating belowground soilborne disease. This is an exciting strategy by which to achieve belowground soilborne disease management by manipulating the aboveground state through aboveground stimulation.

Moisture Controls the Suppression of Panax notoginseng Root Rot Disease by Indigenous Bacterial Communities.

Harnessing indigenous soil microbial suppression is an emerging strategy for managing soilborne plant diseases. Soil moisture is a vital factor in soil microbiomes, but its role in the regulation of microbial suppression is poorly understood. Here, we investigated the correlation of root rot disease of Panax notoginseng with rhizosphere microbial communities mediated by soil moisture gradients from 55% to 100% field capacity (FC); then, we captured the disease-suppressive and disease-inductive microbiomes and validated their functions by a culture experiment with synthetic microbiotas containing keystone species. We found that proper soil moisture at 75% to 95% FC could maintain a disease-suppressive microbiome to alleviate root rot disease. However, extremely low or high soil moistures (>95% FC or <75% FC) could aggravate root rot disease by depleting the disease-suppressive microbiome while enriching the disease-inductive microbiome. Both the low-soil-moisture-enriched pathogen Monographella cucumerina and the high-soil-moisture-enriched pathogen Ilyonectria destructans could synergize with different disease-inductive microbiomes to aggravate disease. Metagenomic data confirmed that low- and high-moisture conditions suppressed antibiotic biosynthesis genes but enriched pathogenicity-related genes, resulting in a change in the soil state from disease suppressive to inductive. This study highlights the importance of soil moisture when indigenous microbial suppression is harnessed for disease control. IMPORTANCE Soilborne diseases pose a major problem in high-intensity agricultural systems due to the imbalance of microbial communities in soil, resulting in the buildup of soilborne pathogens. Harnessing indigenous soil microbial suppression is an emerging strategy for overcoming soilborne plant diseases. In this study, we showed that soil moisture is a key factor in balancing microbiome effects on root rot disease. Proper soil moisture management represent an effective approach to maintain microbial disease resistance by enriching disease-suppressive microbiomes. Conversely, moisture stresses may enrich for a disease-inductive microbiome and aid accumulation of host-specific soilborne pathogens threatening crop production. This work could provide a new strategy for sustainable control of soilborne diseases by enriching the indigenous disease-suppressive microbiome through soil moisture management.

Autotoxin Rg1 Induces Degradation of Root Cell Walls and Aggravates Root Rot by Modifying the Rhizospheric Microbiome.

Management of crop root rot disease is one of the key factors in ensuring sustainable development in agricultural production. The accumulation of autotoxins and pathogens in soil has been reported as a primary driver of root rot diseases; however, less is known about the correlation of plants, their associated pathogens and microbiome mediated by autotoxins as well as the contributions autotoxins make to the occurrence of root rot disease. Here, we integrated metabolomic, transcriptomic, and rhizosphere microbiome analyses to identify the root cell wall degradants cellobiose and d-galacturonic acid as being induced by the autotoxic ginsenoside Rg1 of Panax notoginseng, and we found that exogenous cellobiose and d-galacturonic acid in addition to Rg1 could aggravate root rot disease by modifying the rhizosphere microbiome. Microorganisms that correlated positively with root rot disease were enriched and those that correlated negatively were suppressed by exogenous cellobiose, d-galacturonic acid, and Rg1. In particular, they promoted the growth and infection of the soilborne pathogen Ilyonectria destructans by upregulating pathogenicity-related genes. Cellobiose showed the highest ability to modify the microbiome and enhance pathogenicity, followed by Rg1 and then d-galacturonic acid. Collectively, autotoxins damaged root systems to release a series of cell wall degradants, some of which modified the rhizosphere microbiome so that the host plant became more susceptible to root rot disease. IMPORTANCE The accumulation of autotoxins and pathogens in soil has been reported as a primary driver of root rot disease and one of the key factors limiting sustainable development in agricultural production. However, less is known about the correlation of plants, their associated pathogens, and the microbiome mediated by autotoxins, as well as the contributions autotoxins make to the occurrence of root rot disease. In our study, we found that autotoxins can damage root systems, thus releasing a series of cell wall degradants, and both autotoxins and the cell wall degradants they induce could aggravate root rot disease by reassembling the rhizosphere microbiome, resulting in the enrichment of pathogens and microorganisms positively related to the disease but the suppression of beneficial microorganisms. Deciphering this mechanism among plants, their associated pathogens, and the microbiome mediated by autotoxins will advance our fundamental knowledge of and ability to degrade autotoxins or employ microbiome to alleviate root rot disease in agricultural systems.

Enrichment of Burkholderia in the Rhizosphere by Autotoxic Ginsenosides to Alleviate Negative Plant-Soil Feedback.

The accumulation of autotoxins and soilborne pathogens in soil was shown to be the primary driver of negative plant-soil feedback (NPSF). There is a concerted understanding that plants could enhance their adaptability to biotic or abiotic stress by modifying the rhizosphere microbiome. However, it is not clear whether autotoxins could enrich microbes to degrade themselves or antagonize soilborne pathogens. Here, we found that the microbiome degraded autotoxic ginsenosides, belonging to triterpenoid glycosides, and antagonized pathogens in the rhizosphere soil of Panax notoginseng (sanqi). Deep analysis by 16S rRNA sequencing showed that the bacterial community was obviously changed in the rhizosphere soil and identified the Burkholderia-Caballeronia-Paraburkholderia (BCP) group as the main ginsenoside-enriched bacteria in the rhizosphere soil. Eight strains belonging to the BCP group were isolated, and Burkholderia isolate B36 showed a high ability to simultaneously degrade autotoxic ginsenosides (Rb1, Rg1, and Rd) and antagonize the soilborne pathogen Ilyonectria destructans. Interestingly, ginsenosides could stimulate the growth and biofilm formation of B36, eventually enhancing the antagonistic ability of B36 to I. destructans and the colonization ability in the rhizosphere soil. In summary, autotoxic ginsenosides secreted by P. notoginseng could enrich beneficial microbes in the rhizosphere to simultaneously degrade autotoxins and antagonize pathogen, providing a novel ecological strategy to alleviate NPSF. IMPORTANCE Autotoxic ginsenosides, secreted by sanqi into soil, could enrich Burkholderia sp. to alleviate negative plant-soil feedback (NPSF) by degrading autotoxins and antagonizing the root rot pathogen. In detail, ginsenosides could stimulate the growth and biofilm formation of Burkholderia sp. B36, eventually enhancing the antagonistic ability of Burkholderia sp. B36 to a soilborne pathogen and the colonization of B36 in soil. This ecological strategy could alleviate NPSF by manipulating the rhizosphere microbiome to simultaneously degrade autotoxins and antagonize pathogen.

A Chromosome-Level Genome Assembly of Dendrobium Huoshanense Using Long Reads and Hi-C Data.

Dendrobium huoshanense is used to treat various diseases in traditional Chinese medicine. Recent studies have identified active components. However, the lack of genomic data limits research on the biosynthesis and application of these therapeutic ingredients. To address this issue, we generated the first chromosome-level genome assembly and annotation of D. huoshanense. We integrated PacBio sequencing data, Illumina paired-end sequencing data, and Hi-C sequencing data to assemble a 1.285 Gb genome, with contig and scaffold N50 lengths of 598 kb and 71.79 Mb, respectively. We annotated 21,070 protein-coding genes and 0.96 Gb transposable elements, constituting 74.92% of the whole assembly. In addition, we identified 252 genes responsible for polysaccharide biosynthesis by Kyoto Encyclopedia of Genes and Genomes functional annotation. Our data provide a basis for further functional studies, particularly those focused on genes related to glycan biosynthesis and metabolism, and have implications for both conservation and medicine.

Negative Plant-Soil Feedback Driven by Re-assemblage of the Rhizosphere Microbiome With the Growth of Panax notoginseng.

There is a concerted understanding of the accumulation of soil pathogens as the major driving factor of negative plant-soil feedback (NPSF). However, our knowledge of the connection between plant growth, pathogen build-up and soil microbiome assemblage is limited. In this study, significant negative feedback between the soil and sanqi (Panax notoginseng) was found, which were caused by the build-up of the soil-borne pathogens Fusarium oxysporum, F. solani, and Monographella cucumerina. Soil microbiome analysis revealed that the rhizospheric fungal and bacterial communities were changed with the growth of sanqi. Deep analysis of the phylum and genus levels corroborated that rhizospheric fungal Ascomycota, including the soil-borne pathogens F. oxysporum, F. solani, and especially M. cucumerina, were significantly enriched with the growth of sanqi. However, the bacteria Firmicutes and Acidobacteria, including the genera Pseudomonas, Bacillus, Acinetobacter and Burkholderia, were significantly suppressed with the growth of sanqi. Using microbial isolation and in vitro dual culture tests, we found that most isolates derived from the suppressed bacterial genera showed strong antagonistic ability against the growth of sanqi soil-borne pathogens. Interestingly, inoculation of these suppressed isolates in consecutively cultivated soil could significantly alleviate NPSF. In summary, sanqi growth can suppress antagonistic bacteria through re-assemblage of the rhizosphere microbiome and cause the accumulation of soil-borne pathogens, eventually building negative feedback loops between the soil and plants.

Corrigendum: Tobacco Rotated with Rapeseed for Soil-Borne Phytophthora Pathogen Biocontrol: Mediated by Rapeseed Root Exudates.

[This corrects the article on p. 894 in vol. 7, PMID: 27379037.].

Tobacco Rotated with Rapeseed for Soil-Borne Phytophthora Pathogen Biocontrol: Mediated by Rapeseed Root Exudates.

Black shank, caused by Phytophthora parasitica var. nicotianae, is a widespread and destructive disease of tobacco. Crop rotation is essential in controlling black shank. Here, we confirmed that rotating black shank-infested fields with rapeseed (Brassica napus) suppressed the incidence this disease. Further study demonstrated that rapeseed roots have a strong ability to attract zoospores and subsequently stop the swimming of zoospores into cystospores. Then, rapeseed roots secrete a series of antimicrobial compounds, including 2-butenoic acid, benzothiazole, 2-(methylthio)benzothiazole, 1-(4-ethylphenyl)-ethanone, and 4-methoxyindole, to inhibit the cystospore germination and mycelial growth of P. parasitica var. nicotianae. Thus, rapeseed rotated with tobacco suppresses tobacco black shank disease through the chemical weapons secreted by rapeseed roots.