Characterization of Fusarium solani Associated with Tobacco (Nicotiana tabacum) Root Rot in Henan, China.

The aim of this study was to characterize the Fusarium solani species complex (FSSC) population obtained from tobacco roots with root rot symptoms by morphological characteristics, molecular tests, and assessment of pathogenicity. Cultures isolated from roots were white to cream with sparse mycelium on potato dextrose agar, with colony growth of 21.5 ± 0.5 to 29.5 ± 0.5 mm after 3 days. Sporodochia were cream on carnation leaf agar (CLA) and Spezieller Nährstoffarmer agar (SNA), and macroconidia formed in sporodochia were 3 to 6 septate and straight to slightly curved, with wide central cells, a slightly short blunt apical cell, and a straight to almost cylindrical basal cell with a distinct foot shape, ranging in size from 20.92 to 64.37 × 3.91 to 6.57 µm. Microconidia formed on CLA were reniform and fusiform, with 0 or 1 to occasionally 2 septa, that formed on long monophialidic conidiogenous cells, with a size range of 5.99 to 32.32 × 1.76 to 5.84 µm. Globose to oval chlamydospores were smooth- to rough-walled, 6.5 to 13.3 ± 0.37 µm in diameter, and terminal or intercalary and occurred singly, in pairs, or occasionally in short chains on SNA. Molecular tests consisted of sequencing and phylogenetic analysis of the translation elongation factor-1 alpha (EF-1α), RNA polymerase II largest subunit, and second largest subunit regions. All the obtained sequences revealed 98.14 to 100% identity to F. solani in both Fusarium ID and Fusarium MLST databases. Phylogenetic trees of the EF-1α gene and concatenated three-locus data showed that isolates from tobacco in Henan grouped in the proposed group 5, which is nested within FSSC clade 3 (FSSC 5). Twenty-seven of the 28 isolates caused root rot in artificially inoculated tobacco seedlings, with a disease severity index ranging from 15.00 ± 1.67 to 91.11 ± 2.22. Cross-pathogenicity tests showed that three representative isolates were virulent to six species of Solanaceae and two species of Poaceae, with disease severity indexes ranging from 6.12 ± 0.56 to 84.44 ± 0.00, indicating that these isolates have a wide host range. The results may inform the control of tobacco root rot through improved crop rotations.

Immunomodulatory Protein from Nectria haematococca Induces Apoptosis in Lung Cancer Cells via the P53 Pathway.

Our previous research has shown that a fungal immunomodulatory protein from Nectria haematococca (FIP-nha) possesses a wide spectrum of anti-tumor activities, and FIP-nha induced A549 apoptosis by negatively regulating the PI3K/Akt signaling pathway based on comparative quantitative proteomics. This study further confirmed that the anti-lung cancer activity of FIP-nha was significantly stronger than that of the reported LZ-8 and FIP-fve. Subsequently, 1H NMR-based metabolomics was applied to comprehensively investigate the underlying mechanism, and a clear separation of FIP-nha-treated and untreated groups was achieved using pattern recognition analysis. Four potential pathways associated with the anti-tumor effect of FIP-nha on A549 cells were identified, and these were mainly involved in glycolysis, taurine and hypotaurine metabolism, fructose and mannose metabolism, and glycerolipid metabolism. Metabolic pathway analysis demonstrated that FIP-nha could induce A549 cell apoptosis partly by regulating the p53 inhibition pathway, which then disrupted the Warburg effect, as well as through other metabolic pathways. Using RT-PCR analysis, FIP-nha-induced apoptosis was confirmed to occur through upregulation of p53 expression. This work highlights the possible use of FIP-nha as a therapeutic adjuvant for lung cancer treatment.

Fungal Immunomodulatory Protein from Nectria haematococca Suppresses Growth of Human Lung Adenocarcinoma by Inhibiting the PI3K/Akt Pathway.

Lung cancer is a common disease that is associated with poor prognosis. Fungal immunomodulatory protein from Nectria haematococca (FIP-nha) has potential as a lung cancer therapeutic; as such, illuminating its anti-tumor mechanism is expected to facilitate novel treatment options. Here, we showed that FIP-nha affects lung adenocarcinoma growth ex vivo and in vivo. Comparative quantitative proteomics showed that FIP-nha negatively regulates PI3K/Akt signaling and induces cell cycle arrest, autophagy, and apoptosis. We further demonstrated that FIP-nha suppresses Akt phosphorylation, leading to upregulation of p21 and p27 and downregulation of cyclin B1, cyclin D1, CDK2, and CDK4 expression, ultimately resulting in G1/S and G2/M cell cycle arrest. Meanwhile, FIP-nha-induced PI3K/Akt downregulation promotes A549 apoptosis by increasing the expression ratio of Bax/Bcl-2 and c-PARP and autophagy by decreasing the phosphorylation of mTOR. Thus, we comprehensively revealed the anti-tumor mechanism of FIP-nha, which inhibits tumor growth by modulating PI3K/Akt-regulated cell cycle arrest, autophagy, and apoptosis, and provided the basis for further application of fungal immunomodulatory proteins, especially FIP-nha.

Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani.

Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate knockout mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway for sansalvamide. Sansalvamide is structurally related to the cyclic hexadepsipeptide destruxin, which both contain an α-hydroxyisocaproic acid (HICA) unit. A gene cluster responsible for destruxin production has previously been identified in Metarhizium robertsii together with a hypothetical biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium chrysogenum and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread.

Screening of Endophytic Antagonistic Bacterium from Phellodendron amurense and Their Biocontrol Effects against Canker Rot.

Thirty-four strains of bacteria were isolated from Phellodendron amurense. Using Nectria haematococca as an indicator strain, the best strain, B18, was obtained by the growth rate method. The morphological, physiological and biochemical characteristics of strain B18 and its 16S DNA gene sequence were identified, and the biocontrol effect of strain B18 was assessed in pot and field tests, as well as in a field-control test. Drilling methods were used to determine the antibacterial activity of metabolites from strain B18 and their effects on the growth of pathogen mycelia and spores. The best bacteriostatic rate was 85.4%. B18 can hydrolyse starch and oxidize glucose but does not produce gas; a positive result was obtained in a gelatine liquefaction test. According to 16S DNA gene sequencing, strain B18 is Bacillus methylotrophicus (GenBank accession number: MG457759). The results of pot and field-control trials showed 98% disease control when inoculating 108 cfu/ml of the strain. The disease control effect of the B18 culture liquid (concentrations of 108, 2 × 106, 106, 5 × 105 and 2.5 × 105 cfu/ml) in the field-control test was higher than 80%, and the cure rate of the original delivery solution was 96%. Therefore, in the practical forestry production, a 2.5 × 105 cfu/ml culture liquidshould be applied in advance to achieve good control effects.

COMPARACIÓN DE MÉTODOS DE INOCULACIÓN DE Fusarium solani f. sp. passiflorae EN PLÁNTULAS DE MARACUYÁ (Passiflora edulis f. flavicarpa) / COMPARISON OF METHODS OF INOCULATION OF Fusarium solani f. sp. passiflorae IN SEEDLINGS OF PASSION FRUIT (Passiflora edulis f. flavicarpa)

RESUMEN El maracuyá es una especie susceptible a patógenos, como Fusarium solani f. sp. passiflorae, causante de la secadera, llegando a causar pérdidas entre 90 y 100%, en cultivos comerciales en Colombia. Debido al desconocimiento de un método de inoculación eficiente para evaluar germoplasma de maracuyá contra este patógeno, se planteó este estudio, cuyo objetivo fue comparar cuatro métodos de inoculación de F. solani f. sp. passiflorae MViRi01 en plántulas de maracuyá (Passiflora edulis f. flavicarpa), cultivadas a 29°C, con un fotoperiodo de 12h. Los tratamientos fueron: incisión en el tallo (T1), inmersión de raíces (T2), inyección en el tallo (T3) y cribado en tubos de ensayo modificado (T4). Se estimó la incidencia de la enfermedad y el periodo de incubación del hongo. Los métodos evaluados mostraron diferencias altamente significativas (P<0,001). F. solani f. sp. passiflorae infectó raíces, tallo y hojas, causando estrangulamiento ascendente y descendente del tallo. Se obtuvo 100% de incidencia de la enfermedad con los tratamientos T1 (incisión) y T4 (cribado). En T4, el periodo de incubación del hongo fue 2,4 días y en T2 (inmersión), de 19 días. La muerte de las plántulas ocurrió 12 días después de inocular los tratamientos T1 y T3 (inyección). La emisión de raíces adventicias en las plántulas se indujo inyectando el patógeno (T3). Los métodos apropiados para inocular Fusarium sp. en plántulas de maracuyá fueron el cribado en tubos de ensayo modificado y la incisión en el tallo, debido a la homogeneidad en los resultados y la replicabilidad.

SUMMARY Passionfruit is a species susceptible pathogens such as Fusarium solani f. sp. passiflorae, causal agent of Passiflora collar rot, which can cause yield losses between 90 and 100% in commercial crops in Colombia. Due to lack of knowledge of an efficient inoculation method to evaluate germplasm of passionfruit against this fungus, it was designed this study, whose objective was to compare four methods of inoculation of F. solani f. sp. passiflorae MViRi01 in passionfruit seedlings (Passiflora edulis f. flavicarpa) cultivated at 29ºC and 12-hours of photoperiod. The treatments were: stem incision (T1), root immersion (T2), stem injection (T3), and a modified test-tube screening methodology (T4). Disease incidence and incubation period of the fungus was evaluated. The assessed methods showed highly significant differences (P< 0.001). F. solani f. sp. passiflorae infected roots, stem, and leaves causing stem ascending and descending strangulation. Both T1 (incision) and T4 (screening) treatments showed 100% of disease incidence. The incubation period of the fungus in T4 was 2.4 days and in T2 (immersion) 19 days. The death of the seedlings occurred 12 days after inoculation in T1 and T3 (injection) treatments. The adventitious root sprouting in the seedlings was induced by injecting (T3) the pathogen. The most suitable methods to inoculate F. solani f. sp. passiflorae on passionfruit seedlings were the modified testtube screening methodology and the stem incision due to its homogeneity and replicability results.

Phylogenetic Analysis of Fusarium solani Associated with the Asian Longhorned Beetle, Anoplophora glabripennis.

Culture-independent analysis of the gut of a wood-boring insect, Anoplophora glabripennis (Coleoptera: Cerambycidae), revealed a consistent association between members of the fungal Fusarium solani species complex and the larval stage of both colony-derived and wild A. glabripennis populations. Using the translation elongation factor 1-alpha region for culture-independent phylogenetic and operational taxonomic unit (OTU)-based analyses, only two OTUs were detected, suggesting that genetic variance at this locus was low among A. glabripennis-associated isolates. To better survey the genetic variation of F. solani associated with A. glabripennis, and establish its phylogenetic relationship with other members of the F. solani species complex, single spore isolates were created from different populations and multi-locus phylogenetic analysis was performed using a combination of the translation elongation factor alpha-1, internal transcribed spacer, and large subunit rDNA regions. These analyses revealed that colony-derived larvae reared in three different tree species or on artificial diet, as well as larvae from wild populations collected from three additional tree species in New York City and from a single tree species in Worcester, MA, consistently harbored F. solani within their guts. While there is some genetic variation in the F. solani carried between populations, within-population variation is low. We speculate that F. solani is able to fill a broad niche in the A. glabripennis gut, providing it with fungal lignocellulases to allow the larvae to grow and develop on woody tissue. However, it is likely that many F. solani genotypes could potentially fill this niche, so the relationship may not be limited to a single member of the F. solani species complex. While little is known about the role of filamentous fungi and their symbiotic associations with insects, this report suggests that larval A. glabripennis has developed an intimate relationship with F. solani that is not limited by geographic location or host tree.