The photoactivated antifungal activity and possible mode of action of sodium pheophorbide a on Diaporthe mahothocarpus causing leaf spot blight in Camellia oleifera.

Introduction: Sodium pheophorbide a (SPA) is a natural plant-derived photosensitizer, with high photoactivated antifungal activity against some phytopathogenic fungi. However, its fungicidal effect on Diaporthe mahothocarpus, a novel pathogen that causes Camellia oleifera leaf spot blight, is unclear. Methods: In the present study, we explored its inhibitory effects on spore germination and mycelial growth of D. mahothocarpus. Then we determined its effects on the cell membrane, mycelial morphology, redox homeostasis, and cell death through bioassay. Finally, RNA-seq was used further to elucidate its mode of action at the transcriptional level. Results: We found that SPA effectively inhibited the growth of D. mahothocarpus, with half-maximal effective concentrations to inhibit mycelial growth and spore germination of 1.059 and 2.287 mg/mL, respectively. After 1.0 mg/mL SPA treatment, the conductivity and malondialdehyde content of D. mahothocarpus were significantly increased. Scanning electron microscopy and transmission electron microscopy indicated that SPA significantly affected the morphology and ultrastructure of D. mahothocarpus hyphae, revealing that SPA can destroy the mycelial morphology and cell structure, especially the cell membrane of D. mahothocarpus. Furthermore, transcriptome analysis revealed that SPA significantly suppressed the expression of genes involved in morphology, cell membrane permeability, and oxidative stress. Then, we also found that SPA significantly promoted the accumulation of reactive oxygen species (ROS) in of D. mahothocarpus, while it decreased the content of reduced glutathione, inhibited the enzyme activities of superoxide dismutase and catalase, and exacerbated DNA damage. Annexin V-FITC/PI staining also confirmed that 1.0 mg/mL SPA could significantly induce apoptosis and necrosis. Discussion: Generally, SPA can induce ROS-mediated oxidative stress and cell death, thus destroying the cell membrane and hyphal morphology, and ultimately inhibiting mycelial growth, which indicates that SPA has multiple modes of action, providing a scientific basis for the use of SPA as an alternative plant-derived photoactivated fungicide against C. oleifera leaf spot blight.

Inhibitory effect and mechanism of action of juniper essential oil on gray mold in cherry tomatoes.

Juniper essential oil (JEO), which is mostly known as an immune system booster and effective detoxifier, has substantial antimicrobial activity. A comparison of the inhibitory effects of three plant essential oils from juniper (Juniperus rigida), cedarwood (Juniperus virginiana), and cypress (Crupressus sempervirens) on four plant pathogenic fungi indicated that JEO was the most effective at inhibiting the growth of gray mold (Botrytis cinerea). Additional studies were subsequently conducted to explore the in vivo and in vitro antifungal activity and possible mechanism of JEO against B. cinerea. The results show that JEO inhibited the germination of spores and mycelial growth of B. cinerea in a concentration-dependent manner and exhibited strong inhibition when its concentration exceeded 10 µL/mL. JEO also significantly inhibited the incidence of disease and diameters of gray mold lesions on cherry tomato fruit (Solanum lycopersicum). After 12 h of treatment with JEO, the extracellular conductivity, and the contents of soluble protein, malondialdehyde, and hydrogen peroxide were 3.1, 1.2, 7.2, and 4.7 folds higher than those of the control group, respectively (P < 0.05), which indicated that JEO can damage membranes. Scanning electron microscopy observations revealed that JEO affected the morphology of mycelia, causing them to shrivel, twist and distort. Furthermore, JEO significantly improved the activities of the antioxidant-related enzymes superoxide dismutase and catalase but reduced the pathogenicity-related enzymes polygalacturonase (PG), pectin lyase and endoglucanase of B. cinerea (P < 0.05). In particular, PG was reduced by 93% after treatment with JEO for 12 h. Moreover, the 18 constituents of JEO were identified by gas chromatography/mass spectrometry (GC-MS) analysis, mainly limonene (15.17%), γ-terpinene (8.3%), ß-myrcene (4.56%), terpinen-4-ol (24.26%), linalool (8.73%), α-terpineol (1.03%), o-cymene (8.35%) and other substances with antimicrobial activity. Therefore, JEO can be an effective alternative to prevent and control gray mold on cherry tomato fruit.

First Report of Black Spot Needle Blight of Pinus sylvestris var. mongolica Litv. Caused by Heterotruncatella spartii in China.

Pinus sylvestris var. mongolica Litv. (Pinales: Pinaceae) is an excellent tree for soil and water conservation in Northeast China. The Honghua'erji area in Inner Mongolia is the "hometown of P. sylvestris var. mongolica", however, in recent years, coniferous diseases of P. sylvestris var. mongolica have frequently occurred here. During the investigation, it was found that some black spot needle blight had been observed in addition to the common blight caused by Sphaeropsis sapinea. From May to September 2020, black spot needle blight was found on hundreds of P. sylvestris var. mongolica trees in four forest farms, and the infection rate among the forests was 24.58 % (n=240). This disease first appeared on the upper part of the needles, and the needles then became withered and gradually showed light black spots, although they remained green. As the disease progressed, the needles eventually died and turned gray with many dark black spots. Fungal isolate named YJ-1 was obtained from infected needles of symptomatic pine trees, and a voucher specimen was deposited in Heilongjiang Province Key Laboratory of Forest Protection. Microscopic observation showed the conidia were 3-septate (4 cells) clavate spindles that measured 23.9 µm (20.8-25.9) × 5.9 µm (4.5-8.2) (n=50). The middle two cells were dark brown, and the septa were darker than the cells. Both apical and basal cells were hyaline. The apical cell had 2-4 appendages (mostly 3), and the basal cell had a truncate base (n=50). The cultural characteristics on potato dextrose agar medium were flat off-white and dense in 3-5 d. At approximately 5-7 d, the reverse side of the colony turned pale to slightly luteous. Superficial black acervuli were distributed in the center of the mature colonies after 10 d. Morphological, cultural and microscopic characteristics observed were similar of Heterotruncatella spartii (basionym: Truncatella spartii) reported by Hlaiem et al (2019). To further identify, total DNA was extracted and the internal transcribed spacer region (ITS-rDNA) was amplified by PCR using the primers ITS1/ITS4 and sequenced for BLASTn analysis and phylogenetic tree construction. The resulting 564 bp sequence (GenBank Accession No. OL662864) had 99.24% (521/525) to H. spartii MFLUCC 15-0537, with bootstrap support of at least 94% using the Neighbor-Joining algorithm by MEGA-X (Felsenstein, 1985). The fungus was identified as H. spartii based on morphology and molecular methods. A pathogenicity test was conducted by preparing a conidial suspension of 2.0 × 107 conidia/mL. The suspension was sprayed onto the needles of 20 pots of annual P. sylvestris ar. mongolica seedlings, and the control was sprayed with sterile water. Then the seedlings were placed in a constant temperature room at 25 °C. After 30 d, typical symptoms appeared on 11 inoculated needles, while the control needles remained symptomless. After 50 d, the re-isolation infection rate reached 66.7 %. The fungus present on the inoculated seedlings was morphologically identical to that originally observed on diseased pines, fulfilling Koch's postulates. The fungus was isolated from Spartium junceum for the first time and designated Truncatella spartii (Senanayake et al, 2015). It was then renamed H. spartii (Liu et al, 2019) and has been reported to infect P. pinea in Tunisia (Hlaiem et al, 2019). To our knowledge, this is the first report of H. spartii causing black spot needle blight on P. sylvestris var. mongolica in China and worldwide.

Transcriptomic analysis of interactions between Lymantria dispar larvae and carvacrol.

Due to its biological activity, carvacrol (CAR) is widely used in medicine, agriculture, and forestry. Our previous studies showed that in Lymantria dispar larvae, CAR treatment can induce the production of antifeedants and lead to growth inhibition and death of larvae. However, the effect CAR exerts on RNA levels in L. dispar larvae remains unclear. In this study, the Illumina HiSeq4000 sequencing platform was used to sequence the total RNA of L. dispar larvae. A total of six cDNA libraries (three treatments and three controls) were established and 39,807 genes were generated. Compared with the control group, 296 differentially expressed genes (DEGs) (142 up-regulated and 154 down-regulated) were identified after CAR treatment. GO and KEGG enrichment analyses showed that these DEGs mainly clustered in the metabolism of xenobiotics, carbohydrates, and lipids. Furthermore, 12 DEGs were found to be involved in detoxification, including six cytochrome P450s, two esterases, one glutathione peroxidase, one UDP-glycosyltransferase gene, and two genes encoding heat shock proteins. The expression levels of detoxification genes changed under CAR treatment (especially P450s), which further yielded candidate genes for explorations of the insecticidal mechanism of CAR. The reliability of transcriptome data was verified by qRT-PCR. The enzyme activities of CYP450 and acid phosphatase significantly increased (by 38.52 U/mg·prot and 0.12 µmol/min·mg, respectively) 72 h after CAR treatment. However, the activity of alkaline phosphatase did not change significantly. These changes in enzyme activity corroborated the reliability of the transcriptome data at the protein level. The results of GO enrichment analysis of DEGs indicated that CAR influenced the oxidation-reduction process in L. dispar larvae. Furthermore, CAR can cause oxidative stress in L. dispar larvae, identified through the determination of peroxidase and polyphenol oxidase activities, total antioxidant capacity, and hydrogen peroxide content. This study provides useful insight into the insecticidal mechanism of CAR.

Antifungal Activity of 6-Methylcoumarin against Valsa mali and Its Possible Mechanism of Action.

Valsa canker of apple (VCA) caused by Valsa mali severely affected apple production in east Asia. With the increase in drug resistance, there is an urgent need for efficient and environmentally friendly antifungal agents. Coumarins have attracted much attention due to their excellent antimicrobial activity against plant pathogens. In this study, the antifungal activity of several coumarins against phytopathogenic fungi was evaluated, and then the antifungal activity of the screened 6-MCM against V. mali and its underlying mechanism was further investigated. The results of the in vitro antifungal activity assay showed that some coumarins had significant inhibitory effects on V. mali. Notably, 400 mg/L of 6-MCM had the best antifungal activity of 94.6%. Further experiments showed that 6-MCM slowed down the growth of V. mali mycelia and the germination of spores in a concentration-dependent manner, with EC50 of 185.49 and 54.62 mg/L, respectively. In addition, 6-MCM treatment increased mycelial conductivity, extracellular protein leakage, and MDA content, resulting in damage to the cell membrane. Moreover, 6-MCM significantly reduced the cell wall degrading enzymes secreted by V. mali, including EG, PG and PL, thereby limiting its pathogenic capacity. SEM and TEM results showed that 6-MCM treatment had a significant effect on the morphology and ultrastructure of mycelial cells. Inoculation of isolated apple branches found that the application of 6-MCM effectively inhibited the development of VCA and significantly reduced the incidence. All these results suggest that 6-MCM has the potential as a green substitute for VCA control.

Inhibitory effects and mechanisms of vanillin on gray mold and black rot of cherry tomatoes.

Vanillin is a natural antimicrobial agent; however, there are few reports on its antifungal effect on postharvest pathogenic fungi. This study aimed to investigate the in vivo and in vitro antifungal activities of vanillin against gray mold (caused by B. cinerea) and black rot (caused by A. alternata) of cherry tomato fruit and to explain its possible mechanism of action. Vanillin strongly inhibits Botrytis cinerea and Alternaria alternata mycelial growth, spore germination, and germ tube elongation in a concentration-dependent manner (P<0.05). In vivo experiments showed that 4000 mg L-1 vanillin treatment inhibited cherry tomato gray mold and black rot occurrence. Besides, intercellular electrolytes, soluble proteins, and soluble sugars leakage indicated that 50 or 100 mg L-1 vanillin treatment increased Botrytis cinerea and Alternaria alternata membrane permeability. The increase of malondialdehyde and hydrogen peroxide contents confirmed that 50 or 100 mg L-1 vanillin treatment damages the pathogen membranes. Importantly, vanillin treatment inhibited the pathogenicity-related enzyme activities of the two pathogens to reduce their infection ability, among them PL enzyme activity in A. alternata was most inhibited, reducing by 94.7 % at 6 h treated with 100 mg L-1 vanillin. The hyphae morphology of the two pathogens changed, the mycelia were severely damaged, and the hyphae surface was deformed, shrunk, or even broken after 100 mg L-1 vanillin treatment. In summary, vanillin had a substantial inhibitory effect on postharvest gray mold and black rot in cherry tomato fruit. Therefore, vanillin can be an effective alternative to prevent and control cherry tomato postharvest diseases.

Transcriptome and cell wall degrading enzyme-related gene analysis of Pestalotiopsis neglecta in response to sodium pheophorbide a.

Sodium pheophorbide a (SPA) is a new alternative fungicide with low toxicity and high efficiency, which has high fungicidal activity against Pestalotiopsis neglecta, a pathogen that causes black spot needle blight of Pinus sylvestris var. mongolica. To utilize SPA for plant disease control, understanding its antifungal mechanism is essential. Six cDNA libraries were constructed from 3 d-old P. neglecta mycelia (three SPA-infected and three untreated groups) and 29,850 expressed genes were obtained by Illumina HiSeq4000 sequencing. Compared with controls, 3268 differentially expressed genes (DEGs) were identified in SPA-treated groups, including 1879 upregulated and 1389 downregulated genes. Most DEGs were involved in the metabolism of amino acids, carbohydrates, and lipids, as well as cell structure and genetic information processing. These findings were further confirmed by decreased conductivity, RNA and protein content, and activities of nicotinamide adenine dinucleotide-dependent malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. Moreover, qRT-PCR verified the reliability of the transcriptome results. After treatment with SPA at different concentrations for 60 min, the expressions of three cell wall degrading enzyme-related genes (PnEG, PnBG, and PnPG) were all suppressed. Overall, this study provided insights into the molecular mechanisms through which SPA inhibits P. neglecta, increasing the possibility of developing SPA into an effective fungicide in the future.