Visualization of the Infection and Colonization Process of Dendrobium officinale Using a Green Fluorescent Protein-Tagged Isolate of Fusarium oxysporum.

Dendrobium officinale soft rot is a widespread and destructive disease caused by Fusarium oxysporum that can seriously affect yield and quality. To better understand the fungal infection and colonization, we successfully created an F. oxysporum labeled with green fluorescent protein using the Agrobacterium tumefaciens-mediated transformation method. Transformants had varying fluorescence intensities, but their pathogenicity did not differ from that of the wild type. Fluorescence microscopy revealed that F. oxysporum primarily entered the aboveground portion of D. officinale through the leaf margin, stomata, or by direct penetration of the leaf surface. It then colonized the mesophyll and spread along its vascular bundles. D. officinale exhibited typical symptoms of decay and wilting at 14 days postinoculation, accompanied by a pronounced fluorescence signal in the affected area. The initial colonization of F. oxysporum in the subterranean region primarily involved attachment to the root hair and epidermis, which progressed to the medullary vascular bundle. At 14 days postinoculation, the root vascular bundles of D. officinale exhibited significant colonization by F. oxysporum. Macroconidia were also observed in black rot D. officinale tissue. In particular, the entire root was surrounded by a significant number of chlamydospore-producing F. oxysporum mycelia at 28 days postinoculation. This approach allowed for the visualization of the complete infection process of F. oxysporum and provided a theoretical foundation for the development of field control strategies.

Antifungal activity and mechanism of tetramycin against Alternaria alternata, the soft rot causing fungi in kiwifruit.

Kiwifruit rot caused by the fungus Alternaria alternata occurs in many countries, leading to considerable losses during kiwifruit production. In this study, we evaluated the antifungal activity and mechanism of tetramycin against kiwifruit soft rot caused by Alternaria alternata. Tetramycin exerted antifungal effects through the suppression of mycelial growth, conidial germination, and the pathogenicity of A. alternata. Scanning electron microscopic observations revealed that tetramycin destroyed the mycelial structure, causing the mycelia to twist, shrink, and even break. Furthermore, transmission electron microscopy revealed that tetramycin caused severe plasmolysis and a decrease in cell inclusions, and the cell wall appeared thinner with blurred boundaries. In addition, tetramycin destroyed cell membrane integrity, resulting in the leakage of cellular components such as nucleic acids and proteins in mycelial suspensions. Moreover, tetramycin also caused cell wall lysis by enhancing the activities of chitinase and ß-1,3-glucanase and inducing the overexpression of related chitinase gene (Chit) and ß-1,3-glucanase gene (ß-1,3-glu) in A. alternata. In field trials, tetramycin not only decreased the incidence of kiwifruit rot but also create a beneficial living space for kiwifruit growth. Overall, this study indicated that the application of tetramycin could serve as an alternative measure for the management of kiwifruit rot.

Natural product osthole can significantly disrupt cell wall integrity and dynamic balance of Fusarium oxysporum.

Dendrobium officinale Kimura et Migo is a traditional Chinese herbal medicinal plant. However, the frequent occurrence of soft rot disease (SRD) is one of the most harmful diseases in D. officinale production in recent years, which can seriously affect its yield and quality. In this study, the major pathogenic fungus (SR-1) was isolated from D. officinale with typical symptoms of SRD, and was identified as Fusarium oxysporum through morphological and molecular identification. The biological activities of five natural products were determined against F. oxysporum using a mycelial growth inhibition assay. The results showed that osthole had the highest antifungal activity against F. oxysporum, with an EC50 value of 6.40 mg/L. Scanning electron microscopy (SEM) showed that osthole caused F. oxysporum mycelia to shrink and deform. Transmission electron microscopy (TEM) showed that the organelles were blurred and the cell wall was thickened in the presence of osthole. The sensitivity of F. oxysporum to calcofluor white (CFW) staining was significantly enhanced by osthole. Relative conductivity measurements and propidium iodide (PI) observation revealed that osthole had no significant effect on the cell membrane. Further experiments showed that the activity of chitinase and ß-1,3-glucanase were decreased, and expression levels of chitinase and ß-1,3-glucanase related genes were significantly down-regulated after treatment with osthole. In conclusion, osthole disrupted the cell wall integrity and dynamic balance of F. oxysporum, thereby inhibiting normal mycelial growth.

Repellency Mechanism of Natural Guar Gum-Based Film Incorporated with Citral against Brown Planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

Using of plant essential oil that coevolved as a defense mechanism against agriculture insects is an alternative means of controlling many insect pests. In order to repel brown planthoppers (BPHs), the most notorious rice insect pest, a new film based on guar gum incorporated with citral (GC film) was formulated, which was effective while being environmentally friendly. In this paper, the effect and mechanism of GC film repellency against BPHs were determined. Repellent activity test and olfactory reaction analysis showed that GC film had repellency effect against BPHs, with repellency of 60.00% and 73.93%, respectively. The result of olfactory reaction indicated that GC film repellency against BPHs relied on smell. EPG analysis showed the proportion and mean duration of np waveform were significantly higher than in CK and increased following the treatment concentration, which indicated that GC film affected the recognition of BPHs to rice. Further analysis by RNA sequencing analysis showed a total of 679 genes were significantly upregulated and 284 genes were significantly downregulated in the BPHs fed on the rice sprayed with GC film compared to control. Odorant-binding protein (OBP) gene 797 and gustatory receptor gene (GR)/odorant receptor (OR) gene 13110 showed a significant decrease in differential expression and significant increase in differential expression, respectively. There were 0.66 and 2.55 differential expression multiples between treated BPHs and control, respectively. According to the results described above, we reasoned that GC film repellency against BPHs due to smell, by release of citral, caused the recognition difficulties for BPHs to rice, and OBP gene 797 and GR/OR gene 13110 appeared to be the crucial candidate genes for GC film repellency against BPHs. The present study depicted a clear and consistent repellency effect for GC film against BPHs and preliminarily clarified the mechanism of GC film as a repellent against BPHs, which might offer an alternative approach for control of BPHs in the near future. Our results could also help in the development and improvement of GC films.

Naturally produced magnolol can significantly damage the plasma membrane of Rhizoctonia solani.

Rice sheath blight is a destructive fungal disease caused by Rhizoctonia solani. To find a safe and green measure, the biological activity of six plant extracts against R. solani was determined by mycelial growth rate method. The results showed that magnolol possessed better antifungal activities against R. solani, with an EC50 value of 7.47 mg/L. further action mechanism of magnolol against R. solani was carried out. Studies by scanning electron microscopy (SEM) showed that the morphology of R. solani mycelia was deformation and surface folds. Transmission electron microscope (TEM) observation on treated R. solani showed that magnolol could induce cytoplasmic membrane rupture and cytoplasmic membrane even disappeared completely accompanied with cellular debris was covered around this fungal, and the mycelia treated with magnolol showed fluorescence after PI staining. Further study showed that the content of malondialdehyde (MDA) and activity of chitinase, ß-1,3-glucanase and relative conductivity of mycelia were increased, while the content of soluble protein and activities of catalase (CAT), polyphenol oxidase (PPO), superoxide dismutase (SOD), succinate dehydrogenase (SDH) and NAD-malate dehydrogenase (NAD-MDH) were significantly decreased. These results indicated that magnolol could significantly damage the plasma membrane of R. solani, and interfere with cell respiratory metabolism, thus inhibiting the growth of mycelium.

First Report of Crown Gall of Kiwifruit (Actinidia deliciosa) Caused by Agrobacterium fabacearum in China and the Establishment of Loop-Mediated Isothermal Amplification Technique.

Kiwifruit is moderately sweet and sour and quite popular among consumers; it has been widely planted in some areas of the world. In 2019, the crown gall disease of kiwifruit was discovered in the main kiwifruit-producing area of Guizhou Province, China. This disease can weaken and eventually cause the death of the tree. The phylogeny, morphological and biological characteristics of the bacteria were described, and were related to diseases. The pathogenicity of this species follows the Koch hypothesis, confirming that A. fabacearum is the pathogen of crown gall disease of kiwifruit in China. In this study, Loop-mediated isothermal amplification (LAMP) analysis for genome-specific gene sequences was developed for the specific detection of A. fabacearum. The detection limit of the LAMP method is 5 × 10-7 ng/µL, which has high sensitivity. At the same time, the amplified product is stained with SYBR Green I after the reaction is completed, so that the amplification can be detected with the naked eye. LAMP analysis detected the presence of A. fabacearum in the roots and soil samples of the infected kiwifruit plant. The proposed LAMP detection technology in this study offers the advantages of ease of operation, visibility of results, rapidity, accuracy and high sensitivity, making it suitable for the early diagnosis of crown gall disease of kiwifruit.

Physicochemical Properties and Bioactivity of a New Guar Gum-Based Film Incorporated with Citral to Brown Planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

The brown planthopper (BPH), Nilaparvata lugens (Stål), is the most notorious rice insect pest. In order to repel BPH effectively while being environmentally friendly, a new film based on guar gum incorporated with citral (GC film) was formulated. A toxicity bioassay of citral and guar gum at different proportions (ratios of 3:1, 2:1, 1:1, 1:2, and 1:3 in w/w) of GC film-forming emulsion to BPH was performed with the rice stem dipping method. Results showed that the most effective ratio of citral to guar gum was 1:1 with the median lethal concentration (LC50) of 4.30 mg/mL, far below the LC50 of guar gum (GG)/citral individual (141.51 and 44.38 mg/mL, respectively). The mortality of BPH adults and nymphs in the third instar treated with different dilution multiples of GC film-forming emulsion ranged from 46.67% to 82.22% and from 37.78% to 71.11%, respectively. These indicated that GC film-forming emulsion had a direct toxicity on BPH, and the mixture of citral and GG had synergistic interactions. Subsequently, Fourier-transform infrared spectroscopy showed that the incorporation of guar gum with citral was successful and did not result in the formation of new chemical bonds. The GC film exhibited a darker color and rougher surface topography with larger apertures and deeper gullies (Ra = 1.42 nm, Rq = 2.05 nm, and Rmax = 25.40 nm) compared to the guar gum film (GG film) (Ra = 1.00 nm, Rq = 1.33 nm, and Rmax = 16.40 nm), as determined by transmission electron microscopy and atomic force microscopy. The GC film exhibited a 50.4% lower solubility in water (30.30% vs. 15.00%) and 71.3% oxygen permeability (8.26 × 10-9 vs. 2.37 × 10-9 cm3/m2·d·Pa) (p < 0.05) but did not demonstrate any significant difference in mechanical properties, such as thickness (39.10 vs. 41.70 mm), tensile strength (41.89 vs. 38.30 N/mm2), and elongation at break (1.82% vs. 2.03%) (p < 0.05) compared to the GG film. Our findings established a link between physicochemical properties and bioactivity, which can provide useful information on developing and improving GC films and may offer an alternative approach for the control of BPH in the near future.

Magnolol Loaded on Carboxymethyl Chitosan Particles Improved the Antimicrobial Resistance and Storability of Kiwifruits.

Magnolol is a natural compound extracted from the traditional Chinese medicine Magnolia officinalis, which exhibits antimicrobial properties. However, magnolol is insoluble in water and consists of a phenolic hydroxyl group, which is volatile; these factors hinder its application. In this study, a safe and environmentally friendly method to improve the microbial resistance and storability of harvested fruits is developed using the water-soluble carrier carboxymethyl chitosan (CMCS) and magnolol. Magnolol was loaded on CMCS particles to form Magnolol@CMCS antimicrobial particles, a preservation coating agent. Magnolol@CMCS particles effectively solved the problems of water insolubility and agglomeration of magnolol and reduced the size distribution D50 value of magnolol from 0.749 to 0.213 µm. Magnolol@CMCS particles showed greater toxicity against Staphylococcus aureus, Escherichia coli, and Botryosphaeria dothidea than that of magnolol alone, with effective medium concentration (EC50) values of 0.9408, 142.4144, and 8.8028 µg/mL, respectively. Kiwifruit treated with the Magnolol@CMCS solution showed delayed changes in fruit hardness and soluble solid and dry matter contents and significantly higher ascorbic acid (vitamin C) and soluble total sugar contents and sugar:acid ratios compared with that of the control fruit. In addition, no disease spots were observed on fruit treated with the Magnolol@CMCS solution within 7 days after inoculation with B. dothidea. In conclusion, Magnolol@CMCS particles showed antimicrobial activity on harvested fruits, effectively delayed the hardness and nutritional changes of fruits during storage, and improved the storability of kiwifruit.

A pH-Responsive Essential Oil Delivery System Based on Metal-organic Framework (ZIF-8) for Preventing Fungal Disease.

Smart metal-organic framework nanocarriers that respond to microenvironmental stimuli related to plant diseases can achieve the on-demand release of active ingredients to control diseases. The plant essential oil citral (CT) has significant biological activity against most pathogens but its poor stability limits its application in the field. To improve the applicability of plant essential oils, we aimed to construct a pH responsive essential oil delivery system (CT@ZIF-8) based on the zeolitic imidazolate framework-8 in this study. The high specific surface area of ZIF-8 enables CT@ZIF-8 remarkable loading capacity, and the metal-organic framework effectively delays the volatilization of CT. The results showed that CT@ZIF-8 was approximately 177 nm in size, had a stable nanostructure, and displayed a high pesticide loading efficiency (15.22%). The photodegradation rate of CT loaded in ZIF-8 under UV irradiation (48 h) was only 17.99%, much lower than that of CT alone (51.18%). The CT@ZIF-8 had a pH-responsive controlled release property, releasing CT from CT@ZIF-8 in a pH-dependent manner. The bioactivity results showed that CT@ZIF-8 had a lower EC50 than CT against three fungi (Magnaporthe oryzae, Botryosphaeria dothidea, and Fusarium oxysporum), enhancing the antifungal activity of CT. After 3 d of reagent treatment, only CT@ZIF-8 maintained good control against rice blast (75.76%) and soft rot (63.69%) infection. We have constructed a smart delivery system for essential oils, which provides a new pathway for the efficient application of plant-derived essential oils in the green control of plant diseases.

Natural Product Citronellal can Significantly Disturb Chitin Synthesis and Cell Wall Integrity in Magnaporthe oryzae.

BACKGROUND: Natural products are often favored in the study of crop pests and diseases. Previous studies have shown that citronellal has a strong inhibition effect on Magnaporthe oryzae. The objective of this study was to clarify its mechanism of action against M. oryzae. RESULTS: Firstly, the biological activity of citronellal against M. oryzae was determined by direct and indirect methods, and the results show that citronellal had a strong inhibition effect on M. oryzae with EC50 values of 134.00 mg/L and 70.48 µL/L air, respectively. Additionally, a preliminary study on its mechanism of action was studied. After citronellal treatment, electron microscopy revealed that the mycelium became thin and broken; scanning electron microscopy revealed that the mycelium was wrinkled and distorted; and transmission electron microscopy revealed that the mycelium cell wall was invaginated, the mass wall of mycelium was separated, and the organelles were blurred. The mycelium was further stained with CFW, and the nodes were blurred, while the mycelium was almost non-fluorescent after PI staining, and there was no significant difference in the relative conductivity of mycelium. In addition, chitinase was significantly enhanced, and the expression of chitin synthesis-related genes was 17.47-fold upregulated. Finally, we found that the efficacy of citronellal against the rice blast was as high as 82.14% according to indoor efficacy tests. CONCLUSION: These results indicate that citronellal can affect the synthesis of chitin in M. oryzae and damage its cell wall, thereby inhibiting the growth of mycelium and effectively protecting rice from rice blasts.

Evidences of Colletotrichum fructicola Causing Anthracnose on Passiflora edulis Sims in China.

Passion fruit (Passiflora edulis) is a tropical and subtropical plant that is widely cultivated in China due to its high nutritional value, unique flavor and medicinal properties. In August 2020, typical anthracnose symptoms with light brown and water-soaked lesions on Passiflora edulis Sims were observed, which result in severe economic losses. The incidence of this disease was approximately 30%. The pathogens from the infected fruit were isolated and purified by the method of tissue isolation. Morphological observations showed that the colony of isolate BXG-2 was gray to celadon and grew in concentric circles. The orange conidia appeared in the center after 14 days of incubation. The pathogenicity was verified by Koch's postulates. The internal transcribed spacer (ITS), chitin synthase (CHS-1), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified by relevant PCR programs. The multi-gene (ITS, GAPDH, ACT, CHS-1) phylogeny analysis confirmed that isolate BXG-2 belongs to Colletotrichum fructicola. The inhibitory effect of six synthetic fungicides on the mycelial growth of the pathogen was investigated, among which difenoconazole 10% WG showed the best inhibitory effect against C. fructicola with an EC50 value of 0.5579 mg·L-1. This is the first report of anthracnose on Passiflora edulis Sims caused by Colletotrichum fructicola in China.

Antifungal Activity and Biocontrol Mechanism of Fusicolla violacea J-1 against Soft Rot in Kiwifruit Caused by Alternaria alternata.

Alternaria alternata is the main pathogenic species of various crops, including kiwifruit (Actinidia cinensis). In this study, an antagonistic fungus, J-1, with high antifungal activity against A. alternata was isolated from A. cinensis "Hongyang." The strain J-1 was identified as Fusicolla violacea via morphological identification and DNA sequencing. This study aimed to evaluate the antifungal activity and potential mechanism of the strain J-1 against A. alternata. The strain J-1 exhibited antifungal activity against A. alternata, with an inhibition rate of 66.1% in vitro. Aseptic filtrate (AF) produced by the strain J-1 could suppress the mycelial growth and conidia germination of A. alternata at the inhibition rates of 66.8% and 80%, respectively, as well as suppress the spread of Alternaria rot in fresh kiwifruit. We observed that many clusters of spherical protrusions appeared at the mycelial tips of A. alternata after treatment with 200 mL L-1 AF of J-1. Scanning electron microscopy analysis results showed that the mycelial structures were bent and/or malformed and the surfaces were rough and protuberant. Variations in temperature, pH, and storage time had little effect on the antifungal activity of the AF. Moreover, the AF could damage the integrity of cell membranes and cause intracellular content leakage. Meanwhile, the chitinase and ß-1,3-glucanase enzyme activities increased significantly, indicating that the function of A. alternata cell wall was seriously injured. Eleven antimicrobial metabolites were identified by gas chromatography-mass spectrometry (GC-MS). The strain J-I and its AF exhibited well broad-spectrum antifungal activity against Diaporthe eres, Epicoccum sorghinum, Fusarium graminearum, Phomopsis sp., and Botryosphaeria dothidea, with inhibition rates ranging from 34.4% to 75.1% and 42.7% to 75.2%, respectively. Fusicolla violacea J-1 is a potential biocontrol agent against A. alternata and other fungal phytopathogens.