Bacillus quorum quenching shapes the citrus mycobiome through interkingdom signaling.

Microbiomes are sustained through infinite yet mutually interacting microbial communities, with bacteria and fungi serving as the major constituents. In recent times, microbial interventions have become popular for microbiome manipulation to achieve sustainable goals. Whether and how the introduced biocontrol agent drives fungal microbial assemblages (mycobiome) and the role of interkingdom signaling in shaping the microbiome structure and function remain poorly understood. Here, we implemented wild-type (WT) Bacillus subtilis L1-21 and its quorum quenching (QQ) mutants (L1-21Δytnp, L1-21Δyxel) individually and as consortia to explore the enrichment patterns of key mycobiome members in Huanglongbing (HLB) infected citrus compartments including leaf endosphere, root endosphere, and rhizosphere soil. The application of WT and its QQ mutants produced differential mycobiome enrichment across citrus compartments. Our findings reveal that application of WT B. subtilis enriched beneficial fungi such as Trichoderma (15.82 %) in leaf endosphere. In contrast, pathogenic fungi Fusarium (47.5 %) and Gibberella (0.47 %) involved in citrus root decline were adundant in the L1-21Δytnp treated root endosphere while Nigrospora (11 %) was predominant in L1-21Δyxel treated leaf endosphere, affirming the role of bacterial quorum sensing (QS) molecules in shaping the fungal community composition. In general, based on the fungal functional prediction, fungal pathogens were highly abundant in mutant-treated plants, particularly in leaf endosphere (L1-21Δytnp: 25 %; L1-21Δyxel: 36.35 %) compared to WT (20.93 %). Additionally, some fungal members exhibited strong compartment specificity and both mutants induced distinct mycobiome shifts in rhizosphere soil, leaf, and root endopshere. In conclusion, B. subtilis QQ modifies bacterial QS networks facilitating beneficial fungi to establish, while loss of QQ leads to enrichment of pathogenic fungal groups. Our study provides a direct link of perception and regulation of mycobiome through bacterial-based QS and QQ system, and its association with disease outcomes.

Biocontrol agents transform the stability and functional characteristics of the grape phyllosphere microenvironment.

The spread of grape leaf diseases has a negative impact on the sustainable development of agriculture. Diseases induced by Uncinula necator significantly affect the quality of grapes. Bacillus biocontrol agents have been proven effective in disease management. However, limited research has been conducted on the impact of biocontrol agents on the assembly and potential functions of plant phyllosphere microbial communities. This study used high-throughput sequencing combined with bioinformatics analysis and culture omics technology for analysis. The results showed that biocontrol bacteria B. subtilis utilized in this study can significantly reduce the disease index of powdery mildew (p<0.05); concurrently, it exhibits a lower disease index compared to traditional fungicides. A comprehensive analysis has revealed that biocontrol bacteria have no significant impact on the diversity of phyllosphere fungi and bacteria, while fungicides can significantly reduce bacterial diversity. Additionally, biocontrol agents can increase the complexity of fungal networks and enhance the degree of modularity and stability of the bacterial network. The results also showed that the biocontrol agents, which contained a high amount of B. subtilis, were able to effectively colonize the grapevine phyllosphere, creating a microenvironment that significantly inhibits pathogenic bacteria on grape leaves while enhancing leaf photosynthetic capacity. In conclusion, biocontrol agents significantly reduce the grape powdery mildew disease index, promote a microenvironment conducive to symbiotic microorganisms and beneficial bacteria, and enhance plant photosynthetic capacity. These findings provide a basis for promoting biocontrol agents and offer valuable insights into sustainable agriculture development.

The Response of Murine Gut Microbiome in the Presence of Altered rpoS Gene of Klebsiella pneumoniae.

The murine model is invaluable for studying intricate interactions among gut microbes; hosts; and diseases. However; the impact of genetic variations in the murine microbiome; especially in disease contexts such as Klebsiella pneumoniae (Kp) infection; still needs to be explored. Kp; an opportunistic global pathogen; is becoming increasingly prevalent in regions like Asia; especially China. This study explored the role of the gut microbiota during Kp infection using mouse model; including wild-type and rpoS mutants of Kp138; KpC4; and KpE4 from human; maize; and ditch water; respectively. Under stress conditions; RpoS reconfigures global gene expression in bacteria; shifting the cells from active growth to survival mode. Our study examined notable differences in microbiome composition; finding that Lactobacillus and Klebsiella (particularly in WKp138) were the most abundant genera in mice guts at the genus level in all wild-type treated mice. In contrast; Firmicutes were predominant in the healthy control mice. Furthermore; Clostridium was the dominant genus in all mutants; mainly in ∆KpC4; and was absent in wild-type treated mice. Differential abundance analysis identified that these candidate taxa potentially influence disease progression and pathogen virulence. Functional prediction analysis showed that most bacterial groups were functionally involved in biosynthesis; precursor metabolites; degradation; energy generation; and metabolic cluster formation. These findings challenge the conventional understanding and highlight the need for nuanced interpretations in murine studies. Additionally; this study sheds light on microbiome-immune interactions in K. pneumoniae infection and proposes new potential therapeutic strategies.

Alternaria alternata Pathogen from Cuscuta japonica Could Serve as a Potential Bioherbicide.

Dodder (Cuscuta spp.) is a dangerous parasitic plant that causes serious damage to crop production and is challenging to eliminate. Herbicide application is a common strategy to control dodder in the field, but it is costly, ineffective, and further results in hazardous outcomes. Therefore, our study aims to identify the potential pathogens in naturally occurring dodder infections which may provide efficient biocontrol options. In this regard, the pathogens were isolated from the infected plants, their pathogenicity was validated through inoculation, and the optimal culture conditions for their growth were identified by determining the pathogenicity difference. The pathogenicity range was determined in vitro using the leaves of common horticultural plants and crops. Furthermore, a small range of horticultural plants parasitized by Cuscuta reflexa in the field were inoculated with the pathogen to determine their biosafety and biocontrol potential, and the pathogens were identified by morphological and molecular characterization. We found 7 strains that were isolated after pathogen enrichment culture. Among them, Cbp6 and Cbp7 showed the highest pathogenicity against C. reflexa. After testing the inoculation of more than 50 species of plants, only 9 species showed varying degrees of lesions on leaves, which proved the high biosafety for common plants. Field spraying of these pathogens showed a good control effect on C. reflexa after 21 days; the disease severityreached 66.0%, while its host plant did not display obvious symptoms. In conclusion, the pathogens Cbp6 and Cbp7 were identified as Alternaria alternata, and the results of this study provide a theoretical basis for the biological control of dodder.

The C4 photosynthesis bifunctional enzymes, PDRPs, of maize are co-opted to cytoplasmic viral replication complexes to promote infection of a prevalent potyvirus sugarcane mosaic virus.

In maize, two pyruvate orthophosphate dikinase (PPDK) regulatory proteins, ZmPDRP1 and ZmPDRP2, are respectively specific to the chloroplast of mesophyll cells (MCs) and bundle sheath cells (BSCs). Functionally, ZmPDRP1/2 catalyse both phosphorylation/inactivation and dephosphorylation/activation of ZmPPDK, which is implicated as a major rate-limiting enzyme in C4 photosynthesis of maize. Our study here showed that maize plants lacking ZmPDRP1 or silencing of ZmPDRP1/2 confer resistance to a prevalent potyvirus sugarcane mosaic virus (SCMV). We verified that the C-terminal domain (CTD) of ZmPDRP1 plays a key role in promoting viral infection while independent of enzyme activity. Intriguingly, ZmPDRP1 and ZmPDRP2 re-localize to cytoplasmic viral replication complexes (VRCs) following SCMV infection. We identified that SCMV-encoded cytoplasmic inclusions protein CI targets directly ZmPDRP1 or ZmPDRP2 or their CTDs, leading to their re-localization to cytoplasmic VRCs. Moreover, we found that CI could be degraded by the 26S proteasome system, while ZmPDRP1 and ZmPDRP2 could up-regulate the accumulation level of CI through their CTDs by a yet unknown mechanism. Most importantly, with genetic, cell biological and biochemical approaches, we provide evidence that BSCs-specific ZmPDRP2 could accumulate in MCs of Zmpdrp1 knockout (KO) lines, revealing a unique regulatory mechanism crossing different cell types to maintain balanced ZmPPDK phosphorylation, thereby to keep maize normal growth. Together, our findings uncover the genetic link of the two cell-specific maize PDRPs, both of which are co-opted to VRCs to promote viral protein accumulation for robust virus infection.

Bacillus subtilis YZ-1 surfactins are involved in effective toxicity against agricultural pests.

BACKGROUND: Insect pests negatively affect crop quality and yield. The excessive use of chemical pesticides has serious impacts on the environment and food safety. Therefore, development of effective management strategies in the form of bio-agents have important agricultural applications. Tenebrio molitor, a storage pest, causes losses of grains, medicinal materials, and various agricultural and related products in the warehouse. Bacillus subtilis YZ-1 isolated from naturally deceased Pieris rapae has been found to exhibit significant toxicity against T. molitor. RESULTS: Treatment with B. subtilis YZ-1 fermentation broth resulted in a 90-95% mortality rate of T. molitor within 36 h post-treatment, indicating some active substances may have insecticidal activity in the bacterial supernatant. A bioactivity-guided fractionation method was used to isolate the insecticidal compounds from YZ-1, which led to the identification of surfactins. Additionally, a surfactin deletion mutant YZ-1△srfAA was constructed and the surfactin production by the mutant YZ-1△srfAA was verified through liquid chromatography-mass spectrometry (LC-MS). Further, YZ-1△srfAA exhibited loss of insecticidal activity against T. molitor, Plutella xylostella and Achelura yunnanensis. The insecticidal activity and surfactins contents of several strains of Bacillus sp. were also tested and correlation was found between varying surfactins yield and insecticidal activity exhibited by different strains. CONCLUSION: Conclusively, our results suggest that B. subtilis YZ-1 may provide a novel approach for plant protection against agricultural pests. © 2023 Society of Chemical Industry.

Bacillus amyloliquefaciens AK-12 Helps Rapeseed Establish a Protection against Brevicoryne brassicae.

Aphids are a serious threat to rapeseed (Brassica napus L.) production, and cause unmanageable loss. Therefore, effective prevention and management strategies are urgently required to avoid losses. Bacillus amyloliquefaciens AK-12 isolated from a dead aphid with aphicidal activity was tagged with a green fluorescent protein through a natural transformation. The transformed strains were checked for stability and growth, and the best-performing strain was tested for its colonization inside and outside the rapeseed plant. The stability of AK-12-GFP reached more than 95%, and the growth curve was consistent with that of AK-12. After 30 days of treatment, the colonization of 1 × 106 CFU/g was recorded in rapeseed leaves. Interestingly, AK-12 reduced the aphid transmission rate compared with the control and improved the growth of the rapeseed seedlings. Meanwhile, the AK-12 strain also exhibited phosphorus, potassium-solubilizing, and nitrogen-fixing activity, and produced 2.61 µg/mL of IAA at 24 h. Regulation in the activity of four enzymes was detected after the AK-12 treatment. Phenylalanine ammonia lyase (PAL) was recorded at a maximum of 86.84 U/g after 36 h, and catalase (CAT) decreased after 48 h; however, peroxidase (POD) and polyphenol oxidase (PPO) reached the maximum within 12 h of AK-12 application. Additionally, important resistance genes related to these enzymes were upregulated, indicating the activation of a defense response in the rapeseed against aphids. In conclusion, defense enzymes and defense-related gene activation could improve the pest resistance in rapeseed, which has good application prospects for the future to be developed into biopesticide.

First report of Aloe root and stem rot caused by Phytophthora palmivora in Yunnan Province, China.

Aloe genus plants are perennial evergreen herb belonging to Liliaceae family which is widely used in food, medicine, beauty, and health care (Kumar et al. 2019). In August 2021, symptoms of root and stem rot was observed in approximately 20% of Aloe vera plantings in Yuanjiang County, Yunnan Province, China (23° 64' 53" N, 101° 99' 84" E). The most typical symptoms were stem and root rot, browning and necrosis of vascular tissues, gradual greening, and reddish-browning of leaves from bottom to top, abscission, and eventual plant death (Fig. S1). Therefore, to isolate and identify the pathogen, the plants showing the above symptoms were collected. The plant tissues were cut from the edges of root and stem lesions, followed by disinfection with 75% ethanol for 1 min, rinsed three times with sterilized distilled water, and cut into 3 × 3 mm small squares after excision of marginal tissues. The tissues were transferred to the oomycetes selective medium (Liu et al. 2022) and incubated at 28 °C in the dark for 3~5 days, and suspected colonies were purified. The colonies were then inoculated onto potato dextrose agar (PDA), V8-juice agar (V8), and oatmeal agar (OA) medium plates for morphological characteristics. Finally, 18 isolates with the same colonial and morphological characteristics were obtained from 30 lesioned tissue and one of them was named as ARP1. On PDA, V8 and OA medium plates, the ARP1 colonies were white. On PDA plate, the mycelia were dense and the colonies were petal-like; on V8 plate, the mycelia were cashmere and the colonies were radial or star-like. Whereas, on OA plate, the mycelia were cotton-like and the colonies were fluffy and radial (Fig. S2 A~C). Mycelium did not have septum with high branching and swelling. Sporangia were abundant, semi-papillate, varying in shape from ovoid-ellipsoid to long-ellipsoid, 18-26 × 45-63 µm (average: 22 × 54 µm, n = 30), sporangia released numerous zoospores from the papillate after maturation. The chlamydospores were spherical, 20-35 µm in diameter (average: 27.5 µm, n = 30) (Fig. S2 D~F). These morphological features were like those of the pathogenic species of the oomycetes (Chen et al. 2022). For the molecular characterization, the genomic DNA of the isolate was extracted using the cetyl trimethyl ammonium bromide method, and the translation elongation factor 1α (tef-1α) (Stielow et al. 2015), ß-tubulin (ß-tub) (Kroon et al. 2004) and internal transcribed spacer (ITS) (White et al. 1990) of isolated strain ARP1 were amplified using primer pairs EF1-1018F/EF1-1620R, TUBUF2/TUBUR1 and ITS1/ITS4, respectively. The tef-1α, ß-tub genes and ITS region of ARP1 were directly sequenced and their sequence information was deposited in GenBank under accession numbers OQ506129, OQ506127 and OQ449628. ARP1 was clustered on the same evolutionary branch with Phytophthora palmivora (Fig. S3). To confirm the pathogenicity of ARP1, the main root of A. vera was wounded to 1 cm long and 2 mm deep with a scalpel blade followed by inoculation with 50 ml suspension of ARP1 zoospores at a concentration of 1 × 106 spores / ml per potted plant, and an equal volume of water as control. All inoculated plants were placed in the greenhouse at 28°C, 12 h / 12 h light / dark. After 15 dpi, the inoculated plants showed typical symptoms of wilted and drooping leaves and stem and root rot, same as observed in the field condition (Fig. S4). After inoculation with ARP1, a strain with the same morphological and molecular characteristics as the original isolate was re-isolated, confirming Koch's postulates. To our knowledge, this is the first report of P. palmivora causing root and stem rot of A. vera in the study region. This disease could be a potential risk for aloe production and therefore appropriate management measures should be taken.

Biocontrol of plant pathogens in omics era-with special focus on endophytic bacilli.

Nearly all plants and their organs are inhabited by endophytic microbes which play a crucial role in plant fitness and stress resilience. Harnessing endophytic services can provide effective solutions for a sustainable increase in agriculture productivity and can be used as a complement or alternative to agrochemicals. Shifting agriculture practices toward the use of nature-based solutions can contribute directly to the global challenges of food security and environmental sustainability. However, microbial inoculants have been used in agriculture for several decades with inconsistent efficacy. Key reasons of this inconsistent efficacy are linked to competition with indigenous soil microflora and inability to colonize plants. Endophytic microbes provide solutions to both of these issues which potentially make them better candidates for microbial inoculants. This article outlines the current advancements in endophytic research with special focus on endophytic bacilli. A better understanding of diverse mechanisms of disease control by bacilli is essential to achieve maximum biocontrol efficacy against multiple phytopathogens. Furthermore, we argue that integration of emerging technologies with strong theoretical frameworks have the potential to revolutionize biocontrol approaches based on endophytic microbes.

Complete genome sequence analysis of a novel citlodavirus isolated from the leaves of Myrica rubra in Yunnan.

Through high-throughput sequencing, a novel citlodavirus, tentatively named "Myrica rubra citlodavirus 1" (MRV1, accession no. OP374189), was isolated from the leaves of Myrica rubra in Yunnan exhibiting narrow deformity of leaf tips, shrinkage, and chlorosis along the veins. The complete genome sequence was determined and analyzed using cloning and Sanger sequencing. MRV1 is a single-stranded circular non-enveloped DNA virus with a genome size of 3775 nucleotides and contains six open reading frames (ORFs). The virion-sense genome strand encodes a coat protein (CP, nt 750-1,493, 247 aa), two hypothetical movement proteins (V3, nt 382-666, 94 aa; and V2, nt 461-895, 144 aa), and one movement protein (MP, nt 1,527-2,438, 303 aa). The complementary strand of the genome encodes two replication proteins (RepA, nt 3,712-2,834, 292 aa; Rep, nt 2,867-2,553, 104 aa). The MRV1 genome contains the stem-loop motif 5'-TAATATTAC-3', which is a highly conserved nonanucleotide motif found in the origin of virion-strand replication in geminiviruses. Genome sequence alignment analysis showed that citrus chlorotic dwarf associated virus (CCDaV, accession no. JQ920490) shared the highest nucleotide sequence similarity (66.10% identity) with MRV1. Phylogenetic analysis showed that CCDaV is the closest known relative of MRV1, and that these viruses clustered in a single branch within a clade consisting of citlodaviruses. These results indicate that MRV1 should be regarded as a new species of the genus Citlodavirus in the family Geminiviridae.

Fengycin produced by Bacillus subtilis XF-1 plays a major role in the biocontrol of Chinese cabbage clubroot via direct effect and defense stimulation.

Bacillus subtilis XF-1 is a well-investigated biocontrol agent against the biotrophic Plasmodiophora brassicae Woron., the causal agent of clubroot disease of cruciferous crops. The present study demonstrates that XF-1 could efficiently control clubroot disease via leaf spraying and provides an understanding of the biocontrol mechanisms. High-performance thin-layer chromatography (HTPLC) analysis indicated the presence of fengycin-type cyclopeptides in the supernatant. A ppsB deletion mutant of XF-1 resulted in no fengycin production, significantly reduced the lysis rate of testing spores in vitro and the primary infection rate of root hair in vivo, and decreased the protection value against clubroot disease under the greenhouse conditions. Confocal laser scanning microscopy proved that fengycin was not required for leaf internalization and root colonization. Moreover, the expression level of the ppsB gene in XF-1 was regulated by its cell density in root during interaction with P. brassicae. In addition, the ΔppsB mutant of XF-1 could not efficiently control disease because it led to a lower activation level of the jasmonic acid and salicylic acid signaling pathways in roots, which are necessary for the plant defense reaction upon pathogen invasion. Altogether, the present study provides a new understanding of specific cues in the interaction between B. subtilis and P. brassicae as well as insights into the application of B. subtilis in agriculture.

Environmental effect of agriculture-related manufactured nano-objects on soil microbial communities.

Agriculture-related manufactured nano-objects (MNOs) can revolutionize the crop production and help to achieve sustainable development goals. MNOs with diverse physico-chemical properties and ability to encapsulate and deliver active ingredients in controlled, targeted and stimuli responsive manner can enhance the efficiency while minimizing collateral damage to non-target organisms and environment. Application of MNOs in the form of nanopesticides and nanofertilizers is known to affect soil microbial communities both positively and negatively, but detailed studies with varying dose, type and environmental conditions are scarce. Therefore, it is imperative to understand the complex mechanisms and factors which shape the MNOs-microbial interactions through integrating state of the art technologies including omics (transcriptomics, metabolomics, and proteomics), artificial intelligence, and statistical frameworks. Lastly, we propose the idea of MNOs-mediated manipulation of soil microbiome to modify the soil microbial communities for improved microbial services. These microbial services, if harnessed appropriately, can revolutionize modern agriculture and help in achieving sustainable development goals.

Uniting the Role of Endophytic Fungi against Plant Pathogens and Their Interaction.

Endophytic fungi are used as the most common microbial biological control agents (MBCAs) against phytopathogens and are ubiquitous in all plant parts. Most of the fungal species have roles against a variety of plant pathogens. Fungal endophytes provide different services to be used as pathogen control agents, using an important aspect in the form of enhanced plant growth and induced systemic resistance, produce a variety of antifungal secondary metabolites (lipopeptides, antibiotics and enzymes) through colonization, and compete with other pathogenic microorganisms for growth factors (space and nutrients). The purpose of this review is to highlight the biological control potential of fungal species with antifungal properties against different fungal plant pathogens. We focused on the introduction, biology, isolation, identification of endophytic fungi, and their antifungal activity against fungal plant pathogens. The endosymbionts have developed specific genes that exhibited endophytic behavior and demonstrated defensive responses against pathogens such as antibiosis, parasitism, lytic enzyme and competition, siderophore production, and indirect responses by induced systemic resistance (ISR) in the host plant. Finally, different microscopic detection techniques to study microbial interactions (endophytic and pathogenic fungal interactions) in host plants are briefly discussed.

Novel Virulence Factors Deciphering Klebsiella pneumoniae KpC4 Infect Maize as a Crossing-Kingdom Pathogen: An Emerging Environmental Threat.

Klebsiella pneumoniae is not only a human and animal opportunistic pathogen, but a food-borne pathogen. Cross-kingdom infection has been focused on since K. pneumoniae was identified as the pathogen of maize, banana, and pomegranate. Although the pathogenicity of K. pneumoniae strains (from ditch water, maize, and human) on plant and mice has been confirmed, there are no reports to explain the molecular mechanisms of the pathogen. This study uncovered the K. pneumoniae KpC4 isolated from maize top rot for the determination of various virulence genes and resistance genes. At least thirteen plant disease-causing genes are found to be involved in the disruption of plant defense. Among them, rcsB is responsible for causing disease in both plants and animals. The novel sequence types provide solid evidence that the pathogen invades plant and has robust ecological adaptability. It is imperative to perform further studies on the verification of these KpC4 genes' functions to understand the molecular mechanisms involved in plant−pathogen interactions.

Potential biocontrol efficiency of Trichoderma species against oomycete pathogens.

Plant health is of utmost importance for optimal agricultural production and sustainability. Unfortunately, biotic and abiotic factors put a major constraint on crop safety and productivity. Plant diseases caused by oomycetes inflict serious damage to various crops. Moreover, the injudicious use of chemical pesticides poses threats related to pesticide resistance development in pathogens and environmental pollution. Biocontrol offers an effective solution for disease control; however, research on biocontrol of oomycete-related diseases is scarce. Thus, this study undertakes the screening of biocontrol resources for the effective management of oomycete-related plant diseases. In this regard, 86 isolates of Trichoderma spp. were assessed against Phytophthora nicotianae, P. capsici, Pythium vexans, P. ultimum, and P. dissotocum through dual culture assay. Furthermore, the antagonistic effect of selected isolates was studied against tobacco black shank disease and damping-off of cucumber seedlings in the greenhouse. The relative control effect of the three antagonistic Trichoderma strains AR-4, Tv-1, and ST4-1 on tobacco black shank was more than 60%, which was not significantly different from 6.88 gl-1 fluopicolide-propamocarb. Whereas, the relative control effect of Trichoderma AR-4 and ST4-1 on damping-off of cucumber seedlings was 80.33% and 82.67%, respectively, which were significantly higher than Trichoderma Tv-1 (35.49%) and fluopicolide-propamocarb (47.82%). According to the morphological and molecular characterization, the fungal strains AR-4, Tv-1, and ST4-1 were identified as Trichoderma koningiopsis, T. asperellum, and T. gamsii, respectively. In conclusion, the strains exhibited a strong antagonistic effect against oomycete pathogens and can be integrated into disease management strategies.

Potential of citrus endophyte Bacillus subtilis L1-21 in the control of Candidatus Liberibacter asiaticus in Asian citrus psyllid, Diaphorina citri.

BACKGROUND: Asian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1-21. However, the pathogen elimination and colonization potential of B. subtilis L1-21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown. RESULTS: Initially, we suggested that endophyte L1-21 reduced the CLas copies in ACP from 6.58 × 106 to 5.04 × 104 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium-Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1-21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1-21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1-21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10,4 2.6 × 10,3 and 0 pathogen copies/g fresh midvein, respectively. CONCLUSIONS: We propose that B. subtilis L1-21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP. © 2022 Society of Chemical Industry.

First report of a new leaf blight disease of bletilla (Bletilla striata) caused by Curvularia reesii in China.

Bletilla striata is an important Chinese herbal plant grown widely in southwest China (Qian et al. 2021). Leaf blight was found on cultivated bletilla crops in Yunnan in 2021. The disease infected bletilla leaves and it was present in the field from April to November with the highest incidence (86% plants diseased) recorded in early September in Puer area. Foliar lesions were circular (Φ0.5-1.8 cm) or oval, with pale-gray center and narrow gray-brown outer area surrounded by a yellow halo. The lesions coalesced later to form large irregular spots or blighted areas on leaves. Symptomatic bletilla leaves were sampled from fields in Jiangcheng (E101.8672o, N22.5803o) and Simao (E109.7816o, N22.7891o) counties, Yunnan in July 2021. Seven fungal isolates were obtained from (BJ01-BJ04) and Simao samples (HBJ05-HBJ07) via lesion-tissue culture and hypha-tip purification on PDA medium. A pathogenicity test following Koch's Postulates (Grimms et al. 2006) was conducted using each isolate by inoculating 45-day old bletilla plant (n=30, Zihua cultivar) in a greenhouse through spraying hypha-spore suspension (3.25×104 CFU/mL) prepared with 14 d fresh DNA culture. Non-inoculated plants (n=30) were used as controls. The experiment was repeated once. The isolates BJ02 and HBJ06 (deposited in Yunnan Agric. Univ. Microbes Herbarium) were shown pathogenic to bletilla since similar lesions formed on seedlings 7 d post inoculation and pure fungal cultures with the same colony morphology as those of BJ02 and HBJ06 were re-isolated from leaf lesions 14 dpi. Isolates BJ02 and HBJ06 produced identical colony and conidium morphology after they were incubated at 25oC for 7 d on PDA. Colonies were circular, pale brown, Φ5.5-7.5cm, with villous surface and abundant aerial hyphae. Mycelia were septate, colorless, Φ3-4 µm and with acute-angled branches. Conidiophores developed from hyphae were erect, septate, pale-brown colored and 60-200 µm long. Conidia (produced scarcely and ripened slowly) were long-oval or petaloid, straight or slightly curved, brown, sized 28-45×10-14 µm. Most conidia were divided into 4 cells by 3 septa; the middle two were bigger than the basal and apex cells. Both BJ02 and HBJ06 were identified as Curvularia sp. based on their morphological characters (Tan et al. 2018). The rDNA-ITS, TEF1α and GAPDH genes (Tan et al. 2018) were amplified from these isolates with PCR (White et al. 1990) and sequenced. ITS sequences of the two isolates were both 574 bp (acc. no. OL587997 & OL336480) and 100% (574/574 bp) identical shown by blast comparison. Further blast analyses of ITS (574 bp, OL587997), TEF1α (532 bp, ON637120) and GAPDH (881 bp, ON637121) from isolate BJ02 showed that they were 99.27% (547/551 bp), 100% (842/842 bp) and 99.8% (507/508 bp) identical respectively with those of Curvularia reesii BRIP4358 (MH414907). The 3 genes of BJ02 were concatenated and phylogenic analysis (Tamura et al, 2013) of the concatenated sequence with those of Curvularia spp. showed that BJ02 was clustered with C. reesii BRIP4358 on the same end-branch of the tree with 100% confidence. Therefore, BJ02 and HBJ06 are the same species identified as Curvularia reesii and it is the pathogen causing bletilla leaf blight. C. reesii was first isolated from the air in Australia in 1963 and was named by Tan et al. in 2018. It has not been reported as a plant pathogen elsewhere. This is the first record of this fungus causing bletilla leaf blight in China. Keywords: Bletilla striata; leaf blight; Curvularia reesii; disease symptoms; pathogen morphology; multigene identification References (1) D.J. Grimes. Microbes, 1(5): 223-228, 2006. (2) L.H. Qian et al. Jiangshu Agric. Sci. 49(19): 64-71, 2021. (3) K. Tamura et al. Mol. Bio. & Evol. 30 (12): 2725- 2729, 2013. (4) Y. P. Tan et al. MycoKeys, 35: 1-25. 2018. (5) T.J. White et al. In: PCR Protocols: A Guide to Methods and Applications (eds. M.A. Innis et al.), Acad. Press, Inc. New York. 315-322, 1990.

Phytophthora cinnamomi causing root rot on Rhododendron lapponicum and control it using potential biocontrol agents.

Rhododendron lapponicum (R. lapponicum) is a dwarf Rhododendron species, which is severely infected with root rot and wilt in Yunnan province, China. However, the causal agent causing these symptoms was unknown. An isolate, Pci-1 was identified as Phytophthora cinnamomi, based on its morphology and the sequences of ß-tubulin, internal transcribed spacer, and Ypt1 genes and verified according to the Koch's postulate. We found that this pathogen could infect 14 species of plants, including Althaea rosea, Viburnum cylindricum, and Brassica napus. Strain Pci-1 could cause R. lapponicum to wither and die; and it grows best in an oat medium with pH 7.0 - 8.0 and an optimum temperature of 27°C. We suggest that the rhizosphere of R. lapponicum treated with biocontrol strains Paenibacillus polymyxoides P2-5 and Trichoderma asperellum Tv-1 showed a significant inhibitory effect on pathogen Pci-1. The inhibitory effect of 70% dimethomorph + cymoxanil was significantly higher with EC50 and EC90 values of 0.1894 and 0.3618 a.i. µg/ml, respectively. Greenhouse experiments revealed that the pathogen load is decreased in the presence of potential antagonists. This study provides fundamentals on risk assessment and theoretical support for the management of P. cinnamomi pathogen and contributes significantly to the planting of forest and horticultural crops in a disease-free environment.

Corrigendum: Defeating Huanglongbing Pathogen Candidatus Liberibacter asiaticus With Indigenous Citrus Endophyte Bacillus subtilis L1-21.

[This corrects the article DOI: 10.3389/fpls.2021.789065.].

First report of apple root rot caused by Phytopythium vexans in China.

Apple production is of great economic importance in the fruit industry of China, where Yunnan Province is considered as a major producing area. A survey was conducted to identify apple trees that were problematic from March to November 2020 in Yunnan Province. Symptoms included smaller yellowing leaves, fewer sprouts per branch, browning and necrosis of the roots and lower parts of the stem bark, and wilting. 20% to 45% of apple trees were found infected and randomly scattered in the surveyed orchards. A total of 110 soil samples were collected from the root area of symptomatic apple trees in Tuanjie Town of Kunming City, Zhaotong City, and Malong District of Qujing City in Yunnan Province. Two grams of each soil sample was suspended in 400 ml of sterile water for three days and each soil extract was baited with two apple leaves (Red Fuji's). Following the baiting, those leaves were cut into 10 pieces (5mm×5mm), surface-sterilized with 70% ethanol for 30 seconds, rinsed three times with sterile water, and then air-dried. Each leaf piece was placed in a Petri dish with the oatmeal agar medium containing PCNB 20 mg/ml, rifampicin 20 mg/ml, and then incubated at 25℃ in the dark for 3 days. A mycelial agar plug was picked from the edge of the colonies and transferred to a fresh Potato Dexrose Agar (PDA) plate. Seventy colonies with similar growing characteristics were isolated from the 110 soil samples. Three isolates were retained for further analysis and named XLD8-1, SD1, and YF2. After being cultivated on PDA plates and incubated at 25℃ in the dark for 4 days, their colonies were rose petal-type and white with dense aerial hyphae (Fig 1, A). In ten days of incubation, oogonium measuring 24.55 ± 1.9µm × 20.27 ± 2.3µm and sporangia measuring 21.65 ± 1.3µm × 19.35 ± 1µm were observed (Fig 1, C, D). The total DNA of the isolates was extracted and amplified using three pairs of primers, ITS1/ITS4 (White et al. 1990), LROR/LR7 (LSU) (Vilgalys R, et al. 1990), and FM58/FM66 (COXⅡ) (Martin F N. 2000). The sequences were uploaded to GenBank (Accession No. OL960234, OK037658, OK052604 for ITS, OL960388, OM838413, OM838314 for LSU, and OM962847, OM962848, OM962849 for COXⅡ). ITS sequences of the three isolates (XLD8-1, SD1, YF2) showed 99.87%,99.87%, 99.87% similar to Pp. vexans (Accession No. AB468784, AB468784, and AM701801). LSU sequences of the three isolates showed 99.92%, 99.72%, 100% similar to Pp. vexans (Accession No. EF426541, MT729990, and EF426541). COXⅡ sequences of the three isolates showed 100%, 99.81%, 99.81% similar to Pp. vexans (Accession No. GU133560). Based on the sequence similarity and morphology, the isolates were identified as Phytopythium vexans. Koch's postulates were conducted by wounding the bases of 3 apple seedlings (1-year-old Red Fuji's) with a cork borer. A plug of mycelium of the isolate XLD8-1 grown on PDA plates was placed on each wound (Fig 1, B). Controls were set up to use sterile agar plugs as an inoculum. Seedlings have incubated an incubator at 23-26°C under the alternating light and dark intervals, 12-hours of each. In 15 days, after were inoculated with XLD8-1 the roots and lower part of the stem bark of those seedlings became brownish and necrotic, and their epidermis was easily sloughed off (Fig 1, E-G). The pathogen isolated from the necrotic root tissues were identical to the isolate XLD8-1. Symptoms of apple growth decline caused by Pp. vexans were reported in Morocco (Jabiri Salma, et al. 2021). This experiment verified that Pp. vexans causes root rot of apple. In China, Fusarium sp. is usually considered the main pathogen causing apple root rot. However, the discovery of large numbers of apple trees that were infected by Pp. vexans in Yunnan Province and the confirmation of pathogenicity of Pp. vexans on apple seedlings have demonstrated for the first time that Pp. vexans could cause apple root rot as Fusarium spp does and become an incoming threat to the apple industry, which lays the foundation for study on the disease epidemiology and integrated management of apple root rot in China. References: Jabiri Salma, et al. 2021. Microorganisms, doi:10.3390/MICROORGANISMS9091916. Martin, F. N. 2000. Mycologia, 92(4), 711-727. Vilgalys R., et al. 1990. Journal of Bacteriology, 172:4238-4246 White, T. J., et al. 1990. PCR Protocols: a guide to methods and applications, 18: 315.