Plant Growth Promotion and Biological Control against Rhizoctonia solani in Thai Local Rice Variety "Chor Khing" Using Trichoderma breve Z2-03.

Several strains of Trichoderma are applied in the field to control plant diseases due to their capacity to suppress fungal pathogens and control plant diseases. Some Trichoderma strains also are able to promote plant growth through the production of indole-3-acetic acid (IAA). In southern Thailand, the local rice variety "Chor Khing" is mainly cultivated in the Songkhla province; it is characterized by slow growth and is susceptible to sheath blight caused by Rhizoctonia solani. Therefore, this research aimed to screen Trichoderma species with the ability to promote plant growth in this rice variety and enact biological control against R. solani. A total of 21 Trichoderma isolates were screened for indole compound production using the Salkowski reagent. The Z2-03 isolate reacted positively to the Salkowski reagent, indicating the production of the indole compound. High-performance liquid chromatography (HPCL) confirmed that Z2-03 produced IAA at 35.58 ± 7.60 µg/mL. The cell-free culture filtrate of the potato dextrose broth (CF) of Z2-03 induced rice germination in rice seeds, yielding root and shoot lengths in cell-free CF-treated rice that were significantly higher than those of the control (distilled water and culture broth alone). Furthermore, inoculation with Trichoderma conidia promoted rice growth and induced a defense response against R. solani during the seedling stage. Trichoderma Z2-03 displayed an antifungal capacity against R. solani, achieving 74.17% inhibition (as measured through dual culture assay) and the production of siderophores on the CAS medium. The pot experiment revealed that inoculation with the Trichoderma sp. Z2-03 conidial suspension increased the number of tillers and the plant height in the "Chor Khing" rice variety, and suppressed the percentage of disease incidence (PDI). The Trichoderma isolate Z2-03 was identified, based on the morphology and molecular properties of ITS, translation elongation factor 1-alpha (tef1-α), and RNA polymerase 2 (rpb2), as Trichoderma breve Z2-03. Our results reveal the ability of T. breve Z2-03 to act as a plant growth promoter, enhancing growth and development in the "Chor Khing" rice variety, as well as a biological control agent through its competition and defense induction mechanism in this rice variety.

Biological Control Activities of Rhizosphere Fungus Trichoderma virens T1-02 in Suppressing Flower Blight of Flamingo Flower (Anthurium andraeanum Lind.).

Flower blight caused by Neopestalotiopsis clavispora is an emerging disease of flamingo flower (Anthurium andraeanum Lind.) that negatively impacts flower production. The use of rhizosphere fungi as biocontrol agents is an alternative way to control this disease instead of using synthetic fungicides. This research aimed to screen the potential of rhizosphere fungi, Trichoderma spp., with diverse antifungal abilities to control N. clavispora and to reduce flower blight in flamingo flowers. A total of ten isolates were tested against N. clavispora by dual culture assay, and T1-02 was found to be the most effective isolate against N. clavispora, with inhibition of 78.21%. Morphology and molecular phylogeny of multiple DNA sequences of the genes, the internal transcribed spacer (ITS), translation elongation factor 1-α (tef1-α), and RNA polymerase 2 (rpb2) identified isolate T1-02 as Trichoderma virens. Sealed plate method revealed T. virens T1-02 produced volatile antifungal compounds (VOCs) against N. clavispora, with inhibition of 51.28%. Solid-phase microextraction (SPME) was applied to trap volatiles, and GC/MS profiling showed VOCs emitted from T. virens T1-02 contained a sesquiterpene antifungal compound-germacrene D. The pre-colonized plate method showed that T. virens T1-02 aggressively colonized in tested plates with inhibition of 100% against N. clavispora, and microscopy revealed direct parasitism onto fungal hyphae. Furthermore, the application of T. virens T1-02 spore suspension reduced the disease severity index (DSI) of flower blight in flamingo flowers. Based on the results from this study, T. virens T1-02 displays multiple antagonistic mechanisms and has the potential ability to control flower blight of flamingo flowers caused by N. clavispora.

Electrostatic Atomized Water Particles Induce Disease Resistance in Muskmelon (Cucumis melo L.) against Postharvest Fruit Rot Caused by Fusarium incarnatum.

The postharvest quality of muskmelon can be affected by fruit rot caused by the fungus Fusarium incarnatum, resulting in loss of quality. The utilization of electrostatic atomized water particles (EAWPs) in agriculture applications has been shown to induce disease resistance in plants. Therefore, in this study, we determined the effect of electrostatic atomized water particles (EAWPs) on the disease resistance of muskmelon fruits against postharvest fruit rot caused by F. incarnatum. EAWPs were applied to muskmelon fruits for 0, 30, 60, and 90 min. EAWP-treated muskmelon fruits were inoculated with F. incarnatum, and disease progress was measured. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of the chitinase (CmCHI) and ß-1,3-glucanase (CmGLU) genes of Cucumis melo (muskmelon) was performed for EAWP-treated and -untreated muskmelon fruits. The activities of cell-wall-degrading enzymes (CWDEs), chitinase, and ß-1,3-glucanase were also assayed in EAWP-treated and -untreated muskmelon fruits. The results showed that disease progress was limited by EAWP treatment for 30 min prior to pathogen inoculation. Muskmelon fruits treated with EAWPs for 30 min showed an upregulation of CWDE genes, CmCHI and CmGLU, as observed by qRT-PCR, leading to high chitinase and ß-1,3-glucanase activities, as observed through enzyme assays. The results of SEM microscopy revealed that the effect of the crude enzymes of EAWP-treated muskmelon caused morphological changes in F. incarnatum mycelia. Furthermore, treatment with EAWPs preserved postharvest quality in muskmelon, including with regard to texture stiffness and total chlorophyll contents, compared to untreated muskmelon. These results demonstrate that the pretreatment of muskmelon with EAWPs suppresses the development of F. incarnatum in the early stage of infection by regulating gene expression of CWDEs and elevating the activities of CWDEs, while also maintaining postharvest muskmelon quality.

A New Species of Neoscytalidium hylocereum sp. nov. Causing Canker on Red-Fleshed Dragon Fruit (Hylocereus polyrhizus) in Southern Thailand.

During 2020-2021, cultivated red-fleshed dragon fruit (Hylocereus polyrhizus) in Phatthalung province, southern Thailand, was infected with canker disease in all stages of growth. Small, circular, sunken, orange cankers first developed on the cladodes of H. polyrhizus and later expanded and became gray scabs with masses of pycnidia. The fungi were isolated using the tissue transplanting method and identified based on the growth of the fungal colony, and the dimensions of the conidia were measured. Their species level was confirmed with the molecular study of multiple DNA sequences, and their pathogenicity was tested using the agar plug method. Morphological characterization and molecular identification of the internal transcribed spacer (ITS), translation elongation factor 1-α (tef1-α) and ß-tubulin (tub) sequences revealed the fungal pathogen to be a new species. It was named Neoscytalidium hylocereum sp. nov. The biota of the new species, N. hylocereum, was deposited in Mycobank, and the species was assigned accession number 838004. The pathogenicity test was performed to fulfil Koch's postulates. N. hylocereum showed sunken orange cankers with a mass of conidia similar to those observed in the field. To our knowledge, this is the first report of H. polyrhizus as a host of the new species N. hylocereum causing stem cankers in Thailand.

Morphological and Molecular Characterization of Calonectria foliicola Associated with Leaf Blight on Rubber Tree (Hevea brasiliensis) in Thailand.

Leaf blight is commonly observed in rubber trees (Hevea brasiliensis) and can be caused by several fungal species. From October to December 2021, the emergence rubber tree disease was observed in Krabi province, southern Thailand. Small brown to dark brown spots developed on the leaves of rubber trees and later expanded into most parts of the leaves. Fungal isolates were isolated from infected tissues and a total of 15 Calonectria-like isolates were recovered from 10 infected leaf samples. Pathogenicity testing using the agar plug method revealed that four isolates caused leaf blight on rubber tree, similar to the situation in natural infections. Based on morphological study and the molecular properties of internal transcribed spacer (ITS), calmodulin (cal), translation elongation factor 1-α (tef1-α), and ß-tubulin 2 (tub2) sequences, the four fungal isolates were identified as Calonectria foliicola. To the best of our knowledge, this is the first report of rubber trees pas a new host for C. foliicola in Thailand and elsewhere. This study reports on an emerging disease affecting rubber trees in Thailand, and the results are of benefit for the development of an appropriate method to manage this emerging disease in Thailand.

A Rhizobacterium, Streptomyces albulus Z1-04-02, Displays Antifungal Activity against Sclerotium Rot in Mungbean.

Sclerotium rot causes damping-off and stem rot in seedlings and mature mungbeans, which negatively impacts cultivation. The use of a rhizobacterium to control soil-borne diseases is an alternative method to the excess use of synthetic fungicides; therefore, this study aims to screen rhizosphere actinobacteria with fungicidal activities against Sclerotium rolfsii, the pathogen that causes sclerotium rot in mungbeans. Primary screening showed that the Streptomyces sp. isolate Z1-04-02 displayed the highest effectiveness against S. rolfsii in dual culture plates, with a percentage inhibition of 74.28%. An assay containing enzymes that degrade cell walls, of the cell-free culture filtrate (CF) of Z1-04-02, showed that the activities of chitinase and ß-1,3-glucanase were 0.0209 and 1.0210 U/mL, respectively, which was significantly higher than that of the control (media alone). The cell-free CF of Z1-04-02, incubated at 37 °C and 100 °C, using agar well diffusion, effectively inhibited the growth of S. rolfsii with inhibition percentages of 37.78% and 27.78%, respectively. Solid-phase microextraction (SPME) was applied to trap volatiles released from Z1-04-02 and gas chromatography-mass spectrometry (GC/MS); volatile antifungal compounds were tentatively identified as bicyclic monoterpene (1R)-(-)-myrtenal. The application of the cell-free CF, and the spore suspension of Z1-04-02, showed disease severity indexes (DSIs) of 12.5% and 8.25%, respectively, which were significantly lower than those showing inoculation by S. rolfsii alone. The identification of this strain by morphology, biochemistry tests, and 16s rDNA sequences revealed that Z1-04-02 was Streptomyces albulus. This finding revealed that S. albulus Z1-04-02 displayed diverse fungicidal activities against S. rolfsii, and it has the potential to act as a biological control agent in terms of inhibiting sclerotium rot in mungbeans.

Tobacco Mosaic Virus Infection of Chrysanthemums in Thailand: Development of Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Technique for Sensitive and Rapid Detection.

We detected tobacco mosaic virus (TMV), a member of the genus Tobamovirus and one of the most significant plant-infecting viruses, for the first time in a chrysanthemum in Thailand using reverse-transcription polymerase chain reaction (RT-PCR). The TMV-infected chrysanthemum leaves exhibited mosaic symptoms. We conducted a sequence analysis of the coat protein (CP) gene and found that the TMV detected in the chrysanthemum had 98% identity with other TMV isolates in GenBank. We carried out bioassays and showed that TMV induced mosaic and stunting symptoms in inoculated chrysanthemums. We observed the rigid rod structure of TMV under a transmission electron microscope (TEM). To enhance the speed and sensitivity of detection, we developed a colorimetric RT loop-mediated isothermal amplification (LAMP) technique. We achieved LAMP detection after 30 min incubation in isothermal conditions at 65 °C, and distinguished the positive results according to the color change from pink to yellow. The sensitivity of the LAMP technique was 1000-fold greater than that of RT-PCR, and we found no cross-reactivity with other viruses or viroids. This is the first reported case of a TMV-infected chrysanthemum in Thailand, and our colorimetric RT-LAMP TMV detection method is the first of its kind.

Morphological and Molecular Identification of Plant Pathogenic Fungi Associated with Dirty Panicle Disease in Coconuts (Cocos nucifera) in Thailand.

Dirty panicle disease in coconuts (Cocos nucifera) was first observed in the KU-BEDO Coconut BioBank, Nakhon Pathom province, Thailand. The occurrence of the disease covers more than 30% of the total coconut plantation area. The symptoms include small brown to dark brown spots and discoloration of male flowers. Herein, three fungal strains were isolated from infected samples. Based on the morphological characteristics the fungal isolates, they were classified into two genera, namely, Alternaria (Al01) and Fusarium (FUO01 and FUP01). DNA sequences of internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α (tef1-α), and RNA polymerase II second largest subunit (rpb2) revealed Al01 as Alternaria burnsii, whereas DNA sequences of ITS, rpb2, and tef1-α identified FUO01 and FUP01 as Fusarium clavum and F. tricinctum, respectively. A pathogenicity test by the agar plug method demonstrated that these pathogens cause dirty panicle disease similar to that observed in natural infections. To the best of our knowledge, this is the first report on the novel dirty panicle disease in coconuts in Thailand or elsewhere, demonstrating that it is associated with the plant pathogenic fungi A. burnsii, F. clavum, and F. tricinctum.

Morphological, Molecular Identification and Pathogenicity of Neoscytalidium dimidiatum Causing Stem Canker of Hylocereus polyrhizus in Southern Thailand.

Red-fleshed dragon fruit (Hylocereus polyrhizus) is commonly cultivated in Thailand, especially in southern Thailand, where the weather favors plant growth and development. In 2021, stem canker of H. polyrhizus was observed in a dragon fruit plantation field in Phatthalung Province, southern Thailand. Small, orange circular spots developed on the stem of H. polyrhizus, which later became gray, and the lesion expanded with a mass of conidia. Scytalidium-like fungus was isolated from infected tissues. Based on morphology and phylogenetic analyses of internal transcribed spacer (ITS), nuclear large subunit (LSU) and ß-tubulin (tub) sequences of fungal isolates, the fungus was identified as Neoscytalidium dimidiatum. Pathogenicity tests revealed that this isolate caused stem canker on the stem of H. polyrhizus, similar to that observed in the field. Knowledge of the diagnosis of plant diseases is an important step for managing plant diseases and therefore, this finding provides basic information for the development of appropriate strategies to manage stem canker disease on H. polyrhizus plants.

Trichoderma asperelloides PSU-P1 Induced Expression of Pathogenesis-Related Protein Genes against Gummy Stem Blight of Muskmelon (Cucumis melo) in Field Evaluation.

Gummy stem blight caused by Stagonosporopsis cucurbitacearum is the most destructive disease of muskmelon cultivation. This study aimed to induce disease resistance against gummy stem blight in muskmelon by Trichoderma asperelloides PSU-P1. This study was arranged into two crops. Spore suspension at a concentration of 1 × 106 spores/mL of T. asperelloides PSU-P1 was applied to muskmelon to investigate gene expression. The expression of PR genes including chitinase (chi) and ß-1,3-glucanase (glu) were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and enzyme activity was assayed by the DNS method. The effects of T. asperelloides PSU-P1 on growth, yield, and postharvest quality of muskmelon fruit were measured. A spore suspension at a concentration of 1 × 106 spore/mL of T. asperelloides PSU-P1 and S. cucurbitacearum was applied to muskmelons to determine the reduction in disease severity. The results showed that the expression of chi and glu genes in T. asperelloides PSU-P1-treated muskmelon plants was 7-10-fold higher than that of the control. The enzyme activities of chitinase and ß-1,3-glucanase were 0.15-0.284 and 0.343-0.681 U/mL, respectively, which were higher than those of the control (pathogen alone). Scanning electron microscopy revealed crude metabolites extracted from T. asperelloides PSU-P1-treated muskmelon plants caused wilting and lysis of S. cucurbitacearum hyphae, confirming the activity of cell-wall-degrading enzymes (CWDEs). Application of T. asperelloides PSU-P1 increased fruit weight and fruit width; sweetness and fruit texture were not significantly different among treated muskmelons. Application of T. asperelloides PSU-P1 reduced the disease severity scale of gummy stem blight to 1.10 in both crops, which was significantly lower than that of the control (2.90 and 3.40, respectively). These results revealed that application of T. asperelloides PSU-P1 reduced disease severity against gummy stem blight by overexpressed PR genes and elevated enzyme activity in muskmelon plants.

Volatile Organic Compound from Trichoderma asperelloides TSU1: Impact on Plant Pathogenic Fungi.

Soil microorganisms are well studied for their beneficial effects on plant growth and their impact on biocontrol agents. The production of volatile antifungal compounds emitted from soil fungi is considered to be an effective ability that can be applied in biofumigants in the control of plant diseases. A soil fungus, Trichoderma asperelloides TSU1, was isolated from flamingo flower cultivated soil and identified on the basis of the morphology and molecular analysis of the internal transcribed spacer (ITS), rpb2, and tef1-α genes. To test T. asperelloides TSU1-produced volatile organic compounds (VOCs) with antifungal activity, the sealed plate method was used. The VOCs of T. asperelloides TSU1 inhibited the mycelial growth of fungal pathogens that were recently reported as emerging diseases in Thailand, namely, Corynespora cassiicola, Fusarium incarnatum, Neopestalotiopsis clavispora, N. cubana, and Sclerotium rolfsii, with a percentage inhibition range of 38.88-68.33%. Solid-phase microextraction (SPME) was applied to trap VOCs from T. asperelloides TSU1 and tentatively identify them through gas chromatography-mass spectrometry (GC/MS). A total of 17 compounds were detected in the VOCs of T. asperelloides TSU1, and the dominant compounds were identified as fluoro(trinitro)methane (18.192% peak area) and 2-phenylethanol (9.803% peak area). Interestingly, the commercial 2-phenyethanol showed antifungal activity against fungal pathogens that were similar to the VOCs of T. asperelloides TSU1 by bioassay. On the basis of our study's results, T. asperelloides TSU1 isolated from soil displayed antifungal abilities via the production of VOCs responsible for restricting pathogen growth.

Biocontrol Mechanisms of Trichoderma koningiopsis PSU3-2 against Postharvest Anthracnose of Chili Pepper.

Several mechanisms are involved in the biological control of plant pathogens by the soil-borne Trichoderma spp. fungi. The aim of this study was to characterize a new strain of Trichoderma as a potential biological control agent to control the postharvest anthracnose of chili pepper caused by Colletotrichumgloeosporioides. A total of nine strains of Trichoderma spp. were screened for their antifungal activity using a dual culture assay against C.gloeosporioides. Trichoderma koningiopsis PSU3-2 was shown to be the most effective strain, with a percentage inhibition of 79.57%, which was significantly higher than that of other strains (p < 0.05). In the sealed plate method, T. koningiopsis PSU3-2 suppressed the growth of C.gloeosporioides by 38.33%. Solid-phase microextraction (SPME) was applied to trap volatiles emitted by T. koningiopsis PSU3-2, and the GC/MS profiling revealed the presence of antifungal compounds including azetidine, 2-phenylethanol, and ethyl hexadecanoate. The production of cell-wall-degrading enzymes (CWDEs) was assayed through cell-free culture filtrate (CF) of PSU3-2, and the enzyme activity of chitinase and ß-1,3-glucanase was 0.06 and 0.23 U/mL, respectively, significantly higher than that in the control (p < 0.05). Scanning electron microscopy of the mycelium incubated in cell-free CF of T. koningiopsis PSU3-2 showed the abnormal shape of C.gloeosporioides hyphae. Application of T. koningiopsis PSU3-2 by the dipping method significantly reduced the lesion size (p < 0.05) after inoculation with C.gloeosporioides compared to the control, and there was no disease symptom development in T. koningiopsis PSU3-2-treated chili pepper. This study demonstrates that T. koningiopsis PSU3-2 is an effective antagonistic microorganism and a promising biocontrol agent against postharvest anthracnose of chili pepper, acting with multiple mechanisms.

Postharvest senescent dark spot development mechanism of Musa acuminata ("Khai" banana) peel associated with chlorophyll degradation and stomata cell death.

The occurrence of the postharvest physiological disorder of dark spots on the peel of the ripened "Khai" banana has led to a reduction in its commercial value. The objective of the present study was to investigate the development mechanisms of senescence dark spots of the "Khai" (Musa AA group) banana peel in relation to chlorophyll degradation and stomata cell death. Freshly harvested bananas (commercial mature green stage) were let to ripened at 25 ± 2°C (90%-95% RH). Peel color, senescent spots, DNA degradation, chlorophyll content, chlorophyll-degrading enzyme activities were assessed. The senescent dark spots developed on the ripened bananas right after 6 days of storage, which coincided with remarkably increased DNA degradation, and a rapid decreased of hue angle value and total chlorophyll content which indicated the chlorophyll degradation. The activities of chlorophyllase, chlorophyll-degrading peroxidase and pheophytinase increased gradually to the highest point where the chlorophyll content drastically reduced and the appearance of the dark spots was first recorded after 6 days of storage. These dark spots were observed to be surrounded with a bright luminescent ring of hypermodified fluorescent chlorophyll catabolites (FCCs), the product of chlorophyll breakdown. Additionally, the scanning electron microscope (SEM) revealed that the dark spots were found to have originated from the collapsed cells around the stomata of the ripened banana peel whereby the chlorophyll was entirely diminished. PRACTICAL APPLICATIONS: This research revealed the senescent dark spot development mechanisms of the "Khai" banana peel. The dark spot development symptom on the banana peel surface was caused by the senescence and cell death of the relevant stomata, further associated with chlorophyll degradation. Therefore, any further research into minimizing the dark spot symptom must focus on preventing or delaying stomata senescence and cell death.

Trichoderma asperellum T76-14 Released Volatile Organic Compounds against Postharvest Fruit Rot in Muskmelons (Cucumis melo) Caused by Fusarium incarnatum.

Postharvest fruit rot caused by Fusarium incarnatum is a destructive postharvest disease of muskmelon (Cucumis melo). Biocontrol by antagonistic microorganisms is considered an alternative to synthetic fungicide application. The aim of this study was to investigate the mechanisms of action involved in the biocontrol of postharvest fruit rot in muskmelons by Trichoderma species. Seven Trichoderma spp. isolates were selected for in vitro testing against F. incarnatum in potato dextrose agar (PDA) by dual culture assay. In other relevant works, Trichoderma asperellum T76-14 showed a significantly higher percentage of inhibition (81%) than other isolates. Through the sealed plate method, volatile organic compounds (VOCs) emitted from T. asperellum T76-14 proved effective at inhibiting the fungal growth of F. incarnatum by 62.5%. Solid-phase microextraction GC/MS analysis revealed several VOCs emitted from T. asperellum T76-14, whereas the dominant compound was tentatively identified as phenylethyl alcohol (PEA). We have tested commercial volatile (PEA) against in vitro growth of F. incarnatum; the result showed PEA at a concentration of 1.5 mg mL-1 suppressed fungal growth with 56% inhibition. Both VOCs and PEA caused abnormal changes in the fungal mycelia. In vivo testing showed that the lesion size of muskmelons exposed to VOCs from T. asperellum T76-14 was significantly smaller than that of the control. Muskmelons exposed to VOCs from T. asperellum T76-14 showed no fruit rot after incubation at seven days compared to fruit rot in the control. This study demonstrated the ability of T. asperellum T76-14 to produce volatile antifungal compounds, showing that it can be a major mechanism involved in and responsible for the successful inhibition of F. incarnatum and control of postharvest fruit rot in muskmelons.

Role of Volatiles from the Endophytic Fungus Trichoderma asperelloides PSU-P1 in Biocontrol Potential and in Promoting the Plant Growth of Arabidopsis thaliana.

Fungal volatile organic compounds (VOCs) emitted by Trichoderma species interact with a plant host and display multifaceted mechanisms. In this study, we investigated the antifungal activity of VOCs emitted by Trichoderma asperelloides PSU-P1 against fungal pathogens, as well as the ability of VOCs to activate defense responses and to promote plant growth in Arabidopsis thaliana. The strain's VOCs had remarkable antifungal activity against fungal pathogens, with an inhibition range of 15.92-84.95% in a volatile antifungal bioassay. The VOCs of T. asperelloides PSU-P1 promoted the plant growth of A. thaliana, thereby increasing the fresh weight, root length, and chlorophyll content in the VOC-treated A. thaliana relative to those of the control. High expression levels of the chitinase (CHI) and ß-1,3-glucanase (GLU) genes were found in the VOC-treated A. thaliana by quantitative reverse transcription polymerase chain reaction (RT-PCR). The VOC-treated A. thaliana had higher defense-related enzyme (peroxidase (POD)) and cell wall-degrading enzyme (chitinase and ß-1,3-glucanase) activity than in the control. The headspace VOCs produced by PSU-P1, trapped with solid phase microextraction, and tentatively identified by gas chromatography-mass spectrometry, included 2-methyl-1-butanol, 2-pentylfuran, acetic acid, and 6-pentyl-2H-pyran-2-one (6-PP). The results suggest that T. asperelloides PSU-P1 emits VOCs responsible for antifungal activity, for promoting plant growth, and for inducing defense responses in A. thaliana.

The 2b protein of cucumber mosaic virus is essential for viral infection of the shoot apical meristem and for efficient invasion of leaf primordia in infected tobacco plants.

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Delta 2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Delta 2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Delta 2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Delta 2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Delta 2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Delta 2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Delta 2b, whereas accumulation of Delta 2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.