Antifungal effects of volatile organic compounds produced by Trichoderma koningiopsis T2 against Verticillium dahliae.

Volatile organic compounds (VOCs) produced by microorganisms are considered promising environmental-safety fumigants for controlling soil-borne diseases. Verticillium dahliae, a notorious fungal pathogen, causes economically important wilt diseases in agriculture and forestry industries. Here, we determined the antifungal activity of VOCs produced by Trichoderma koningiopsis T2. The VOCs from T. koningiopsis T2 were trapped by solid-phase microextraction (SPME) and tentatively identified through gas chromatography-mass spectrometry (GC/MS). The microsclerotia formation, cell wall-degrading enzymes and melanin synthesis of V. dahliae exposed to the VOC mixtures and selected single standards were examined. The results showed that the VOCs produced by strain T2 significantly inhibited the growth of V. dahliae mycelium and reduced the severity of Verticillium wilt in tobacco and cotton. Six individual compounds were identified in the volatilome of T. koningiopsis T2, and the dominant compounds were 3-octanone, 3-methyl-1-butanol, butanoic acid ethyl ester and 2-hexyl-furan. The VOCs of strain T2 exert a significant inhibitory effect on microsclerotia formation and decreased the activities of pectin lyase and endo-ß-1,4-glucanase in V. dahliae. VOCs also downregulated the VdT3HR, VdT4HR, and VdSCD genes related to melanin synthesis by 29. 41-, 10. 49-, and 3.11-fold, respectively. Therefore, T. koningiopsis T2 has potential as a promising biofumigant for the biocontrol of Verticillium wilt disease.

Identification of Bacillus velezensis SBB and Its Antifungal Effects against Verticillium dahliae.

Traditional control methods have drawbacks in controlling Verticillium wilt diseases caused by Verticillium dahliae Kleb.; therefore, an efficient and environmentally friendly strategy for disease control must be identified and the mechanisms determined. In this study, a soil-isolated strain SBB was identified as Bacillus velezensis based on 16S rRNA, gyrA, and gyrB gene sequences. In vitro, strain SBB had excellent inhibitory effects on V. dahliae, with the highest inhibition rate of 70.94%. Moreover, strain SBB inhibited production of the conidia of V. dahliae and suppressed the production of microsclerotia and melanin. Through gas chromatograph-mass spectrometer analysis, nine compounds were detected from the volatile organic compounds produced by SBB, among which 2-nonanol, 2-heptanone, 6-methyl-2-heptanone, and 2-nonanone could completely inhibit V. dahliae growth. Strain SBB produced cellulase, amylase, protease, and siderophore. During inhibitory action on V. dahliae, strain SBB showed upregulated expression of genes encoding non-volatile inhibitory metabolites, including difficidin, bacilysin, and bacillaene, at 1.923-, 1.848-, and 1.448-fold higher, respectively. Thus, our study proved that strain SBB had an efficient antagonistic effect on V. dahliae, suggesting strain SBB can be used as a potential biological control agent against Verticillium wilt.

First Report of Diaporthe eres Causing Leaf Spot on Pseudocydonia sinensis in China.

Pseudocydonia sinensis is a Chinese ornamental plant with great landscaping value. Its fruit is additionally used for medicinal purposes (Lim 2012). In June 2020, a leaf spot disease was observed in the campus of Nanjing Forestry University (32°04'34.53″N 118°48'42.06″E). The symptoms began with irregular red-brown spots, which gradually enlarged, extended and overlapped, with an incidence of 85% (29/34 trees). Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of tissues. Pure cultures were obtained by monosporic isolation.The representative isolate NJMG 5-7 was used for morphological and molecular characterization. The growing fungal colony on PDA was initially white, but gradually turned grey. Pycnidia formation was observed on PDA supplemented with alfalfa stems. The pycnidia produced two different types of conidia, α and ß, which ooze out in yellow creamy mucilaginous masses. Conidiophores were hyaline, cylindrical and smooth, 16.8 to 33.1 × 1.5 to 2.6 µm (n=30). Conidiogenous cells were 13.6 to 29.3 × 1.5 to 2.7 µm (n=30). The α-conidia were, unicellular, hyaline elliptical or fusiform, bi-guttulate, 6.5 to 9.2 × 1.8 to 3.3 µm (n = 50). The ß-conidia were hyaline, aseptate, without guttules, filiform, curved, with obtuse ends, 12.5 to 25 × 1.0 to 1.8 µm (n = 50). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, and calmodulin (CAL), translation elongation factor 1 alpha (EF1-a), and beta-tubulin genes (TUB) were amplified from isolate NJMG 5-7 with primers ITS1/ITS4 (White et al. 1990), CAL-228F/CAL-737R (Carbone & Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (OP223050 for ITS, OP252809 for CAL, OP252807 for EF1-a, and OP252808 for TUB). A BLAST search of GenBank showed that ITS, CAL, EF1-a and TUB sequences of NJMG 5-7 were similar to those of D. eres CBS 138594 (99% identity), AR5193 (99%), AR5193 (99%) and MG281193 (100%), respectively. The morphological and molecular results identified the isolate as D. eres (Feng et al. 2015). To fulfill Koch's postulates, a pathogenicity test was conducted using three P. sinensis plants. Six leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of D. eres from a 3-day-old culture grown on PDA. Inoculations with sterile PDA plugs on different leaves of the same tree were used as controls. All inoculated leaves were enclosed in plastic bags together with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags to keep moisture conditions. Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and identified from its morphology, thus fulfilling Koch's postulates. This fungus can cause a variety of diseases. To our knowledge, this is the first report of D. eres causing leaf spots on P. sinensis in the world. These findings provide a foundation for future studies on the epidemiology and control of this newly emerging disease.

Rahnella aquatilis JZ-GX1 alleviates iron deficiency chlorosis in Cinnamomum camphora by secreting desferrioxamine and reshaping the soil fungal community.

Plant growth-promoting rhizobacteria are important for improving plant iron nutrition, but the interactions among inoculants, host plants and soil microorganisms have not been greatly explored. Rahnella aquatilis JZ-GX1 was applied to treat the increasingly serious iron deficiency chlorosis in Cinnamomum camphora, and the resulting improvement in chlorosis was determined by assessing the contents of chlorophyll, active iron, Fe2+ and antioxidant enzymes in leaves, the effects on the soil microbial community and the metabolism in the rhizosphere by high-throughput sequencing techniques and liquid chromatography-mass spectrometry (LC-MS). The results showed that inoculation with JZ-GX1 significantly increased the chlorophyll content of C. camphora, which promoted the redistribution of active iron in roots and leaves, increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), and thus reduced membrane damage in iron-deficient C. camphora caused by reactive oxygen species. According to genome prediction and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis, the JZ-GX1 strain could secrete desferrioxamine (DFO), and the concentration of DFO in C. camphora rhizosphere was 21-fold higher than that in uninoculated soil. The exogenous application of DFO increased the SPAD and Fe2+ contents in leaves. In addition, the inoculant affected the fungal community structure and composition in the C. camphora rhizosphere soil and increased the abundances of specific taxa, such as Glomus, Mortierella, Trichoderma, and Penicillium. Therefore, R. aquatilis JZ-GX1 application promoted iron absorption in C. camphora trees by secreting DFO and alleviated iron deficiency chlorosis through interactions with the local fungal community.

Physiological and Transcriptome Analyses Revealed the Mechanism by Which Deferoxamine Promotes Iron Absorption in Cinnamomum camphora.

Iron deficiency causes chlorosis and growth inhibition in Cinnamomum camphora, an important landscaping tree species. Siderophores produced by plant growth-promoting rhizobacteria have been widely reported to play an indispensable role in plant iron nutrition. However, little to date has been determined about how microbial siderophores promote plant iron absorption. In this study, multidisciplinary approaches, including physiological, biochemical and transcriptome methods, were used to investigate the role of deferoxamine (DFO) in regulating Fe availability in C. camphora seedlings. Our results showed that DFO supplementation significantly increased the Fe2+ content, SPAD value and ferric-chelate reductase (FCR) activity in plants, suggesting its beneficial effect under Fe deficiency. This DFO-driven amelioration of Fe deficiency was further supported by the improvement of photosynthesis. Intriguingly, DFO treatment activated the metabolic pathway of glutathione (GSH) synthesis, and exogenous spraying reduced glutathione and also alleviated chlorosis in C. camphora. In addition, the expression of some Fe acquisition and transport-related genes, including CcbHLH, CcFRO6, CcIRT2, CcNramp5, CcOPT3 and CcVIT4, was significantly upregulated by DFO treatment. Collectively, our data demonstrated an effective, economical and feasible organic iron-complexing agent for iron-deficient camphor trees and provided new insights into the mechanism by which siderophores promote iron absorption in plants.

Effects of Volatile Organic Compounds Produced by Pseudomonas aurantiaca ST-TJ4 against Verticillium dahliae.

Verticillium dahliae is one of the most destructive fungal pathogens, causing substantial economic losses in agriculture and forestry. The use of plant growth-promoting rhizobacteria (PGPR) is an effective and environmentally friendly strategy for controlling diseases caused by V. dahliae. In this study, 90 mm in diameter Petri plates were used to test the effect of volatile organic compounds (VOCs) produced by different concentrations of Pseudomonasaurantiaca ST-TJ4 cells suspension on V. dahliae mycelia radial growth and biomass. The mycelial morphology was observed by using scanning electron microscopy. The conidia germination and microsclerotia formation of V. dahliae were evaluated. The VOCs with antifungal activity were collected by headspace solid-phase microextraction (SPME), and their components were analyzed by gas chromatography-mass spectrometry (GC-MS). The VOCs produced by strain ST-TJ4 significantly inhibited the growth of mycelium of V. dahliae. The morphology of the hyphae was rough and wrinkled when exposed to VOCs. The VOCs of strain ST-TJ4 have a significant inhibitory effect on V. dahliae conidia germination and microsclerotia formation. At the same time, the VOCs also reduce the expression of genes related to melanin synthesis in V. dahliae. In particular, the expression of the hydrophobin gene (VDAG-02273) was down-regulated the most, about 67-fold. The VOCs effectively alleviate the severity of cotton root disease. In the volatile profile of strain ST-TJ4, 2-undecanone and 1-nonanol assayed in the range 10-200 µL per plate revealed a significant inhibitory effect on V. dahliae mycelial radial growth. These compounds may be useful to devise new control strategies for control of Verticillium wilt disease caused by V. dahliae.

First Report of Botryosphaeria dothidea Causing Leaf Blight on Cornus officinalis in Jiangsu Province, China.

Cornus officinalis Sieb. et Zucc., belonging to the family Cornaceae, is often used as an ornamental plant and is widely distributed in Shandong, Jiangsu, and Zhejiang provinces and other places in China. Since 2020, a new disease with high incidence has been found in Xuanwu Lake Park (32°04'34.53″N 118°48'42.06″E) in Nanjing, Jiangsu Province, China. The symptoms began as small brown lesions formed along the leaf tips, which gradually expanded and became dark brown with a light brown border. A survey of C. officinalis trees in Xuanwu Lake Park showed that approximately 90% of thirty trees were infected, which decreased the ornamental value of C. officinalis. Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated in 90% of the tissues. Pure cultures were obtained by monosporic isolation. A representative isolate, SZY 2-2, was used for morphological and molecular characterization. The colonies were initially white, gradually turning gray green to black with copious gray aerial mycelium after 1 week in culture. Conidia were one-celled, hyaline, smooth, and fusoid to ellipsoid. Conidia measurements were 23.6±1.9×7.2±0.56 µm (n = 50). The morphology of SZY 2-2 matched the description of Botryosphaeria dothidea (Slippers et al. 2004). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-a) gene, and beta-tubulin gene (TUB), were amplified from isolate SZY 2-2 with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Koho 1999), and ßt2a/ßt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (ON171471 for ITS, ON185540 for EF1-a, and ON185541 for TUB). A BLAST search of GenBank showed that ITS, EF1-a and TUB sequences of SZY 2-2 were similar to those of B. dothidea MN633360 (identity=517/517 bp; 100%), MK783294 (identity=299/299 bp; 100%), and KF005081 (identity=461/461 bp; 100%), respectively. The morphological and molecular results identified the isolate as B. dothidea (Zhai et al. 2014). To fulfill Koch's postulates, a pathogenicity test was conducted using three C. officinalis plants. Five leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of B. dothidea from a 5-day-old culture grown on PDA, and inoculation with sterile PDA plugs on different leaves of the same tree served as negative controls. The leaves were enclosed in plastic bag along with the branches with a wet cotton ball inside. Sterile H2O2 was sprayed into the plastic bags to keep moisture conditions.Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and had morphology as characterized before, thus fulfilling Koch's postulates. B. dothidea is known as a ubiquitous fungus and operates as both an endophyte and an opportunistic pathogen of trees (Slippers and Wingfield 2007, Zhao et al 2020). Stress factors that predispose trees to disease expression by B. dothidea include drought, defoliation (Theodore et al. 1997), competition, and physical damage (Slippers and Wingfield 2007). This is consistent with the occurrence of the disease in September and association of B. dothidea with the presence of wounds. More investigation is needed to determine the relationship between possible endophytic growth of B. dothidea on C. officinalis and the leaf blight found in Jiangsu Province.

Genome Sequencing of Rahnella victoriana JZ-GX1 Provides New Insights Into Molecular and Genetic Mechanisms of Plant Growth Promotion.

Genomic information for bacteria within the genus Rahnella remains limited. Rahnella sp. JZ-GX1 was previously isolated from the Pinus massoniana rhizosphere in China and shows potential as a plant growth-promoting (PGP) bacterium. In the present work, we combined the GridION Nanopore ONT and Illumina sequencing platforms to obtain the complete genome sequence of strain JZ-GX1, and the application effects of the strain in natural field environment was assessed. The whole genome of Rahnella sp. JZ-GX1 comprised a single circular chromosome (5,472,828 bp, G + C content of 53.53%) with 4,483 protein-coding sequences, 22 rRNAs, and 77 tRNAs. Based on whole genome phylogenetic and average nucleotide identity (ANI) analysis, the JZ-GX1 strain was reidentified as R. victoriana. Genes related to indole-3-acetic acid (IAA), phosphorus solubilization, nitrogen fixation, siderophores, acetoin, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, gamma-aminobutyric acid (GABA) production, spermidine and volatile organic compounds (VOCs) biosynthesis were present in the genome of strain JZ-GX1. In addition, these functions were also confirmed by in vitro experiments. Importantly, compared to uninoculated control plants, Pyrus serotina, Malus spectabilis, Populus euramericana (Dode) Guinier cv. "San Martino" (I-72 poplar) and Pinus elliottii plants inoculated with strain JZ-GX1 showed increased heights and ground diameters. These findings improve our understanding of R. victoriana JZ-GX1 as a potential biofertilizer in agriculture.

Improvement of Sphaeropsis Shoot Blight Disease Resistance by Applying the Ectomycorrhizal Fungus Hymenochaete sp. Rl and Mycorrhizal Helper Bacterium Bacillus pumilus HR10 to Pinus thunbergii.

Ectomycorrhizal fungi (EMFs) form symbioses with plant roots to promote nutrient uptake by plants but it is controversial as to whether they induce disease resistance in plants. Here, we inoculated pine seedlings with Sphaeropsis sapinea, which was presymbiotic with the EMF Hymenochaete sp. Rl, and the mycorrhizal helper bacterium (MHB) Bacillus pumilus HR10, which promotes the formation of Pinus thunbergia-Hymenochaete sp. Rl mycorrhizae. The results showed that inoculation with Hymenochaete sp. Rl, B. pumilus HR10, and the consortium significantly reduced pine shoot blight disease caused by S. sapinea. After inoculation with pathogenic fungi, callose deposition was significantly increased in needles of pine seedlings inoculated with Hymenochaete sp. Rl, B. pumilus HR10, and the consortium, together with an increase in enzymatic and nonenzymatic systemic antioxidant activity as well as early priming for upregulated expression of PR3 and PR5 genes. Our findings suggest that ectomycorrhizal colonization enhances the resistance of pine seedlings to Sphaeropsis shoot blight by triggering a systemic defense response and that interactions between EMFs and MHBs are essential for mycorrhizal-induced disease resistance.

Inhibitory Effects of Phenazine Compounds and Volatile Organic Compounds Produced by Pseudomonas aurantiaca ST-TJ4 Against Phytophthora cinnamomi.

Phytophthora cinnamomi is an important plant pathogen that is widely distributed worldwide and has caused serious ecological damage and significant economic losses in forests and plantations in many countries. The use of plant growth-promoting rhizobacteria is an effective and environmentally friendly strategy for controlling diseases caused by P. cinnamomi. In this study, we investigated the antagonistic mechanism of Pseudomonas aurantiaca ST-TJ4 against P. cinnamomi through different antagonistic approaches, observations of mycelial morphology, study of mycelial metabolism, and identification of antagonistic substances. The results showed that Pseudomonas aurantiaca ST-TJ4 was able to significantly inhibit mycelial growth, causing mycelial deformation and disrupting internal cell structures. Additionally, pathogen cell membranes were damaged by ST-TJ4, and mycelial cell content synthesis was disrupted. Ultraperformance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses showed that phenazine compounds and 2-undecanone were the main antagonistic components. The ammonia produced by the ST-TJ4 strain also contributed to the inhibition of the growth of P. cinnamomi. In conclusion, our results confirm that Pseudomonas aurantiaca ST-TJ4 can inhibit P. cinnamomi through multiple mechanisms and can be used as a biological control agent for various plant diseases caused by P. cinnamomi.

Colonization by the Mycorrhizal Helper Bacillus pumilus HR10 Is Enhanced During the Establishment of Ectomycorrhizal Symbiosis Between Hymenochaete sp. Rl and Pinus thunbergii.

There are complex interactions between mycorrhizal helper bacteria (MHBs) and ectomycorrhizal (ECM) fungi, with MHBs promoting mycorrhizal synthesis and ECM fungi regulating plant rhizobacterial colonization, diversity, and function. In this study, to investigate whether the ECM fungus Hymenochaete sp. Rl affects the survival and colonization of the MHB strain Bacillus pumilus HR10 in the rhizosphere, the biomass of B. pumilus HR10 was measured in the rhizosphere and mycorrhizosphere. In addition, extracts of Hymenochaete sp. Rl and Pinus thunbergii were evaluated for their effect on B. pumilus HR10 colonization (growth, sporulation, biofilm formation, extracellular polysaccharide and extracellular protein contents, flagellar motility, and expression of colonization-related genes). The results showed that inoculation of Hymenochaete sp. Rl significantly increased the biomass of B. pumilus HR10 in the rhizosphere; however, while extracts of Hymenochaete sp. Rl and P. thunbergii did not affect the biomass or spore formation of HR10, they did affect its biofilm formation, extracellular polysaccharide and extracellular protein production, and flagellar motility. Furthermore, the addition of symbiont extracts affected the expression of chemotaxis-related genes in HR10. When the extracts were added separately, the expression of srf genes in HR10 increased; when the extracts were added simultaneously, the expression of the flagellin gene fliG in HR10 increased, but there was no significant effect on the expression of srf genes, consistent with the results on biofilm production. Thus, Hymenochaete sp. Rl and P. thunbergii roots had a positive effect on colonization by B. pumilus HR10 at the rhizosphere level through their secretions.

Fine Identification and Classification of a Novel Beneficial Talaromyces Fungal Species from Masson Pine Rhizosphere Soil.

Rhizosphere fungi have the beneficial functions of promoting plant growth and protecting plants from pests and pathogens. In our preliminary study, rhizosphere fungus JP-NJ4 was obtained from the soil rhizosphere of Pinus massoniana and selected for further analyses to confirm its functions of phosphate solubilization and plant growth promotion. In order to comprehensively investigate the function of this strain, it is necessary to ascertain its taxonomic position. With the help of genealogical concordance phylogenetic species recognition (GCPSR) using five genes/regions (ITS, BenA, CaM, RPB1, and RPB2) as well as macro-morphological and micro-morphological characters, we accurately determined the classification status of strain JP-NJ4. The concatenated phylogenies of five (or four) gene regions and single gene phylogenetic trees (ITS, BenA, CaM, RPB1, and RPB2 genes) all show that strain JP-NJ4 clustered together with Talaromyces brevis and Talaromyces liani, but differ markedly in the genetic distance (in BenA gene) from type strain and multiple collections of T. brevis and T. liani. The morphology of JP-NJ4 largely matches the characteristics of genes Talaromyces, and the rich and specific morphological information provided by its colonies was different from that of T. brevis and T. liani. In addition, strain JP-NJ4 could produce reduced conidiophores consisting of solitary phialides. From molecular and phenotypic data, strain JP-NJ4 was identified as a putative novel Talaromyces fungal species, designated T. nanjingensis.

Identification of Two Fungal Pathogens Responsible for Liriodendron chinense × tulipifera Black Spot and Screening of Trichoderma sp. for Disease Control.

Liriodendron chinense × tulipifera black spot is a newly discovered disease that causes yellowing and early shedding of leaves, affecting the growth of Liriodendron trees, and significantly reducing their ornamental value as a garden species. The pathogen responsible for this disease, and how it can be prevented and controlled, are not clear. In this study, the occurrence of this disease was first investigated according to Koch's postulates, and the primary pathogens causing Liriodendron black spot were determined to be Colletotrichum gloeosporioides and Alternaria alternata. Biocontrol strains antagonistic to these two pathogens were then screened from the leaf microorganisms of L. chinense × tulipifera, and a preliminary investigation of the biological control of Liriodendron black spot was performed. Through the screening of antagonistic microorganisms on the leaf surface of L. chinense × tulipifera, the strain Trichoderma koningiopsis T2, which displayed strong antagonism against C. gloeosporioides and A. alternata, was obtained. The T2 strain could inhibit the growth of the two pathogens via three mechanisms: hyperparasitism, volatile and nonvolatile metabolite production, and environmental acidification. The biocontrol experiments in the greenhouse and field showed that initial spraying with a T. koningiopsis T2 spore suspension followed by the two pathogens resulted in the lowest disease incidence. These results confirmed the black spot pathogens of L. chinense × tulipifera, clarified the antagonistic mechanism of T. koningiopsis T2 against the two pathogens, and provided a theoretical basis and technical support for the biological control of the disease.

Volatile Organic Compounds of the Plant Growth-Promoting Rhizobacteria JZ-GX1 Enhanced the Tolerance of Robinia pseudoacacia to Salt Stress.

Salt stress is one of the major abiotic stresses that affects plant growth and development. The use of plant growth-promoting rhizobacteria to mitigcate salt stress damage in plants is an important way to promote crop growth under salt stress conditions. Rahnella aquatilis JZ-GX1 is a plant growth-promoting rhizobacterial strain, but it is not clear whether it can improve the salt tolerance of plants, and in particular, the role of volatile substances in plant salt tolerance is unknown. We investigated the effects of volatile organic compounds (VOCs) from JZ-GX1 on the growth performance, osmotic substances, ionic balance and antioxidant enzyme activities of acacia seedlings treated with 0 and 100mm NaCl and explored the VOCs associated with the JZ-GX1 strain. The results showed that compared to untreated seedlings, seedlings exposed to plant growth-promoting rhizobacterium JZ-GX1 via direct contact with plant roots under salt stress conditions exhibited increases in fresh weight, lateral root number and primary root length equal to approximately 155.1, 95.4, and 71.3%, respectively. Robinia pseudoacacia seedlings exposed to VOCs of the JZ-GX1 strain showed increases in biomass, soil and plant analyser development values and lateral root numbers equal to 132.1, 101.6, and 166.7%, respectively. Additionally, decreases in malondialdehyde, superoxide anion (O2 -) and hydrogen peroxide (H2O2) contents and increases in proline contents and superoxide dismutase, peroxidase and glutathione reductase activities were observed in acacia leaves. Importantly, the sodium-potassium ratios in the roots, stems, and leaves of acacia exposed to VOCs of the JZ-GX1 strain were significantly lower than those in the control samples, and this change in ion homeostasis was consistent with the upregulated expression of the (Na+, K+)/H+ reverse cotransporter RpNHX1 in plant roots. Through GC-MS and creatine chromatography, we also found that 2,3-butanediol in the volatile gases of the JZ-GX1 strain was one of the important signaling substances for improving the salt tolerance of plants. The results showed that R. aquatilis JZ-GX1 can promote the growth and yield of R. pseudoacacia under normal and salt stress conditions. JZ-GX1 VOCs have good potential as protectants for improving the salt tolerance of plants, opening a window of opportunity for their application in salinized soils.

Enhanced Iron Uptake in Plants by Volatile Emissions of Rahnella aquatilis JZ-GX1.

Iron deficiency in soil has crucially restricted agricultural and forestry production. Volatile organic compounds (VOCs) produced by beneficial microorganisms have been proven to play an important role in inducing abiotic stress tolerance in plants. We investigated the effects of VOCs released by the rhizobacterium Rahnella aquatilis JZ-GX1 on the growth and root parameters of Arabidopsis thaliana under iron deficiency. The effect of the rhizobacterial VOCs on the gene expression in iron uptake and hormone signaling pathways were detected by RT-qPCR. Finally, the VOCs of the JZ-GX1 strain that could promote plant growth under iron deficiency stress were screened. The results showed that the JZ-GX1 strain could induce A. thaliana tolerance to iron deficiency stress by promoting the development of lateral roots and root hairs and increasing the activities of H+ ATPase and Fe3+ reductase. In addition, the AHA2, FRO2, and IRT1 genes of A. thaliana exposed to JZ-GX1-emitted VOCs were upregulated 25-, 1. 81-, and 1.35-fold, respectively, and expression of the abscisic acid (ABA) synthesis gene NCED3 was upregulated on both the 3rd and 5th days. Organic compounds were analyzed in the headspace of JZ-GX1 cultures, 2-undecanone and 3-methyl-1-butanol were found to promote Medicago sativa and A. thaliana growth under iron-limited conditions. These results demonstrated that the VOCs of R. aquatilis JZ-GX1 have good potential in promoting iron absorption in plants.

Phytase-Producing Rahnella aquatilis JZ-GX1 Promotes Seed Germination and Growth in Corn (Zea mays L.).

Phytase plays an important role in crop seed germination and plant growth. In order to fully understand the plant growth-promoting mechanism by Rahnella aquatilis JZ-GX1, the effect of this strain on germination of maize seeds was determined in vitro, and the colonization of maize root by R. aquatilis JZ-GX1 was observed by scanning electron microscope. Different inoculum concentrations and Phytate-related soil properties were applied to investigate the effect of R. aquatilis JZ-GX1 on the growth of maize seedlings. The results showed that R. aquatilis JZ-GX1 could effectively secrete indole acetic acid and had significantly promoted seed germination and root length of maize. A large number of R. aquatilis JZ-GX1 cells colonized on the root surface, root hair and the root interior of maize. When the inoculation concentration was 107 cfu/mL and the insoluble organophosphorus compound phytate existed in the soil, the net photosynthetic rate, chlorophyll content, phytase activity secreted by roots, total phosphorus concentration and biomass accumulation of maize seedlings were the highest. In contrast, no significant effect of inoculation was found when the total P content was low or when inorganic P was sufficient in the soil. R. aquatilis JZ-GX1 promotes the growth of maize directly by secreting IAA and indirectly by secreting phytase. This work provides beneficial information for the development and application of R. aquatilis JZ-GX1 as a microbial fertilizer in the future.

Salt Tolerance Mechanism of the Rhizosphere Bacterium JZ-GX1 and Its Effects on Tomato Seed Germination and Seedling Growth.

Salinity is one of the strongest abiotic factors in nature and has harmful effects on plants and microorganisms. In recent years, the degree of soil salinization has become an increasingly serious problem, and the use of plant growth-promoting rhizobacteria has become an option to improve the stress resistance of plants. In the present study, the salt tolerance mechanism of the rhizosphere bacterium Rahnella aquatilis JZ-GX1 was investigated through scanning electron microscopy observations and analysis of growth characteristics, compatible solutes, ion distribution and gene expression. In addition, the effect of JZ-GX1 on plant germination and seedling growth was preliminarily assessed through germination experiments. R. aquatilis JZ-GX1 was tolerant to 0-9% NaCl and grew well at 3%. Strain JZ-GX1 promotes salt tolerance by stimulating the production of exopolysaccharides, and can secrete 60.6983 mg/L of exopolysaccharides under the high salt concentration of 9%. Furthermore, the accumulation of the compatible solute trehalose in cells as the NaCl concentration increased was shown to be the primary mechanism of resistance to high salt concentrations in JZ-GX1. Strain JZ-GX1 could still produce indole-3-acetic acid (IAA) and siderophores and dissolve inorganic phosphorus under salt stress, characteristics that promote the ability of plants to resist salt stress. When the salt concentration was 100 mmol/L, strain JZ-GX1 significantly improved the germination rate, germination potential, fresh weight, primary root length and stem length of tomato seeds by 10.52, 125.56, 50.00, 218.18, and 144.64%, respectively. Therefore, R. aquatilis JZ-GX1 is a moderately halophilic bacterium with good growth-promoting function that has potential for future development as a microbial agent and use in saline-alkali land resources.

First Report of leaf spot disease caused by Colletotrichum gloeosporioides on Chaenomeles sinensis in China.

Chaenomeles sinensis is a shrub or small arbor of the genus Chaenomeles in Rosaceae, which is widely planted in China. It is a kind of garden ornamental plant and has high economic value. Since 2020, a leaf disease occurred on the foliage of C. sinensis at the campus of Nanjing Forestry University, Nanjing, China. After investigating, C. sinensis was found with leaf spot disease at a 100% infection rate, which causing gigantic ornamental loss. Leaf spots are round to irregular distributing on the leaves, in addition, the color of spots is brown. There are yellow halos on the edge of the lesion. Small leaf tissues (3 to 4 mm2) from lesion margins were surface sterilized with 75% ethanol for 30s and then rinsed with sterile dH2O for three times. Afterwards, placed on potato dextrose agar (PDA) at 25°C. Pure cultures were obtained by monosporic isolation, and a representative isolate (NJTJ.1) was obtained. When cultured on PDA, the colony of NJTJ.1 was white and cottony. On the reverse side, the color of colony nearly light yellow. The colony were placed in the liquid Carboxymethyl cellulose (CMC) medium. After culturing for 24h in a shaker at 25℃ and 150rmp/min, the spore liquid was taken by us. The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and measured 15.1 to 23.6× 5.4 to 7.9 µm (n =30). Appressoria were one-celled, brown, thick-walled, ellipsoidal, and measured 7.7 to 13.8 × 6.4 to 10.3 µm (n =30). The morphological characteristics of NJTJ.1 fitted with the description of the Colletotrichhum gloeosporioides complex (Weir et al., 2012). For accurate identification, the internal transcribed spacer (ITS), and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS-1) were amplified with primers ITS1/ITS4, GDF/GDR, ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al, 2019), respectively. The sequences were deposited in GenBank [Accession Nos.MT984264, MW030495 and MW030496 to MW030497 for NJTJ.1]. A Blast search of GenBank showed that ITS, GAPDH, ACT and CHS-1 sequences of NJTJ.1 were 99%, 99%, 100% and 100% identical to those of C. gloeosporioides (MH571757.1 ,KY995355.1 , MN058143.1 and MN313581.1). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate NJTJ.1 clustered in the C. gloeosporioides clade with 99% bootstrap support. The pathogenicity of the NJTJ.1 was verified both on detached and living leaves. The detached leaves were inoculated with 5-mm mycelial plugs cut from the edge of 6-day old cultures on PDA and 20 µL of spore suspension (106 conidia/mL) and each treatment had 5 replicates. Controls were treated with sterile dH2O. The inocula were placed at a distance of 2 to 3 cm on the leaves which were wounded with a sterile needle. All of them were placed in 20-cm dishes on wet filter paper at 25°C. After 5 days, all the inoculated points showed lesions which were similar to those outdoor observed. Whereas, controls were asymptomatic.At the same time, the plugs of C. gloeosporioides were inoculated on living leaves.After 7 days, the leaves which were inoculated also appeared lesions. This is the first report of C. gloeosporioides causing leaf blotch on Chaenomeles sinensis in China. These data will help develop effective strategies for managing this disease.

Identification, cloning and expression patterns of the genes related to phosphate solubilization in Burkholderia multivorans WS-FJ9 under different soluble phosphate levels.

As important plant growth-promoting rhizobacteria, phosphate-solubilizing bacteria (PSB) fix nitrogen, dissolve potassium, promote growth, improve the soil micro-environment, and enhance soil fertility. A high-efficiency PSB strain from the pine tree rhizosphere, Burkholderia multivorans WS-FJ9, was screened in our laboratory. In this study, using a Bio Screener fully automatic microbial growth curve meter to determine the growth of the WS-FJ9 strain in phosphate-removing medium, the growth and mineral phosphate solubilization of WS-FJ9 were measured by Mo-Sb colorimetry and organophosphate-solubilization plate assays. Second-generation sequencing technology was used to obtain genomic information and to analyze possible phosphate decomposition genes. The related expression levels of these genes under different soluble phosphate levels were determined by quantitative real-time PCR. The results showed that WS-FJ9 had strong adaptability and capacity for mineral phosphate solubilization at low soluble phosphate levels, which is characterized by its low soluble phosphate induction and high soluble phosphate inhibition. The amount of solubilized mineral phosphate could exceed 140 mg/L. The total length of the WS-FJ9 genome was 7,497,552 bp after splicing, and the GC content was 67.37%. Eight phosphate-related genes were selected to determine their expression patterns at different soluble phosphate levels. Among them, AP-2, GspE and GspF were only related to organic phosphate, HlyB was only related to inorganic phosphate, and PhoR, PhoA, AP-1 and AP-3 were related to both. The WS-FJ9 strain utilizes multiple pathways for mineral phosphate solubilization, and the solubilization processes of different phosphate sources are interrelated and independent, indicating that the WS-FJ9 strain can adapt to different phosphate source environments and has good potential for future applications.

Antifungal Effects of Volatile Organic Compounds Produced by Rahnella aquatilis JZ-GX1 Against Colletotrichum gloeosporioides in Liriodendron chinense × tulipifera.

The use of volatile organic compounds (VOCs) produced by microorganisms for the biological control of plant diseases has attracted much attention in recent years. In this study, the antifungal activity and identity of VOCs produced by Rahnella aquatilis JZ-GX1 isolated from the rhizosphere soil of pine were determined and analyzed. The effect of the VOCs on the mycelial growth of Colletotrichum gloeosporioides, the pathogen of Liriodendron chinense × tulipifera black spot, was determined by a joined-petri dish fumigation method. An in vitro leaf inoculation method was used to determine the fumigation effect of the VOCs on Liriodendron black spot. VOCs with antifungal activity were collected by headspace solid-phase microextraction (SPME), and their components were analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that the VOCs secreted by JZ-GX1 inhibited the mycelial growth of the tested pathogen. The VOCs destroyed the morphology of the mycelium, significantly increased the permeability of the cell membrane and downregulated the expression of pathogenicity-related genes during mycelial infection, thus inhibiting the expansion of anthracnose disease spots in leaves. In the volatile compound profile, 3-methyl-1-butanol and 2-phenylethyl methyl ether significantly inhibited the mycelial growth and spore germination of C. gloeosporioides. This work provides a new strategy for the research and application of microorganisms and bioactive compounds to control plant anthracnose.