RESUMO
Hazelnut is among the most important nut crops in Chile, currently covering 46,000 ha. In 2023, the country exported 30,000-ton. In recent years the incidence of plants with internal discoloration, cankers and dieback has been increasing. In some cases, the trees died and had to be removed and, after a year, purple resupinate fruiting bodies were observed growing from the stumps. To determine the etiology of the symptoms and signs, wood samples (n=318) were collected since 2020, from 38 symptomatic orchards from Maule to La Araucanía Regions, primarily from the cvs. Tonda di Giffoni and Lewis. Wood sections 0.5 cm diameter were cut from the symptomatic tissues, disinfected using a sodium hypochlorite (10%) solution, and plated on a quarter-strength acidified potato dextrose agar (aPDA1/4). The plates were incubated and purified on PDA. Subsequently, isolates were identified by morphological and molecular means. Almost half of the isolates (47%) were preliminarily identified as basidiomycetes, based on mycelial features such as the presence of clamp connections, with 45% of them exhibiting abundant whitish cottony fast-growth mycelia, resembling Chondrostereum purpureum (Grinbergs et al., 2020). DNA was extracted and the 500-bp fragment, located between 5S and 18S ribosomal regions, was amplified using APN1 specific primers (Becker et al. 1999), identifying the isolates as C. purpureum. In addition, 5.8S gene of RGM1 (35°13'40.9"S 71°25'14.1"W), RGM2 (36°31'27.95"S 71°46'58.31"W), RGM3 (37°10'54.8"S 72°03'39.6"W), RGM4 (35°19'25.2"S 71°19'54.7"W) and RGM5 (36°35'30.8"S 72°05'18.8"W) isolates, representing different locations within the hazelnut growing area, was amplified using ITS1/ITS4 primers (White et al., 1990). The PCR product was sequenced, and the analysis showed 100% homology among isolates (Genebank codes: PP839283, PP839284, PP839285, PP839286 and PP839287, respectively). To determine the pathogenicity of the isolates, 30-cm healthy cuttings cv. Lewis were inoculated with mycelial plugs, while control shoots were inoculated with sterile agar plugs. Cuttings were vertically arranged in pots with 3-cm water and incubated for 60-d at 22°C. In addition, fresh cuts of 3-y potted plants cv. Lewis were inoculated with mycelial plugs and incubated for 137-d in a shadehouse. After incubation, bark was removed from inoculated cuttings and the length of necrotic lesions was measured. Although discoloration was reproduced by all the isolates in both pathogenicity tests, RGM1 isolate was the most aggressive, causing the complete discoloration of the cuttings and the death of the inoculated plants. To our knowledge this is the first report of C. purpureum causing wood disease in hazelnut. These findings are significant because the disease may not only reduce orchard longevity but also decrease fruit yield and quality, as observed in other fruit crops (Grinbergs et al., 2021).
RESUMO
Pectinases are widely used in a variety of industrial processes. However, their application is limited by low catalytic processivity, reduced stability, high cost, and poor re-use compatibility. These drawbacks may be overcome by enzyme immobilization with ferromagnetic nanoparticles, which are easily recovered by a magnetic field. In this work, an endopolygalacturonase from Chondrostereum purpureum (EndoPGCp) expressed in Pichia pastoris was immobilized on glutaraldehyde-activated chitosan ferromagnetic nanoparticles (EndoPGCp-MNP) and used to supplement a commercial enzyme cocktail. No significant differences in biochemical and kinetic properties were observed between EndoPGCp-MNP and EndoPGCp, although the EndoPGCp-MNP showed slightly increased thermostability. Cocktail supplementation with EndoPGCp-MNP increased reducing sugar release from orange wastes by 1.8-fold and showed a synergistic effect as compared to the free enzyme. Furthermore, EndoPGCp-MNP retained 65% of the initial activity after 7 cycles of re-use. These properties suggest that EndoPGCp-MNP may find applications in the processing of pectin-rich agroindustrial residues.
Assuntos
PoligalacturonaseRESUMO
Silverleaf is an important fungal trunk disease of fruit crops, such as Japanese plum (Prunus salicina). It is known that infection by Chondrostereum purpureum results in discolored wood, "silvered" foliage, and tree decline. However, effects on fruit yield and quality have not been assessed. Therefore, the objectives of this study were to determine C. purpureum pathogenicity on P. salicina and the effects on physiology, fruit yield, and quality, in Chile, in 2019 and 2020. Wood samples from affected plum trees were collected in the Chilean plum productive area. Fungi were isolated by plating wood sections from the necrosis margin on culture media. Morphological and molecular characteristics of the isolates corresponded to C. purpureum (98%). Representative isolates were inoculated from healthy plum plants and after 65-d incubation, wood necrotic lesions and silver leaves were visible. Fungi were reisolated, fulfilling Koch's postulates. To determine Silverleaf effects, xylem water potential and fruit yield and quality were measured in healthy and Silverleaf-diseased plum trees 'Angeleno'. Water potential was altered in diseased trees, and fruit yield was reduced by 51% (2019) and by 41% (2020) compared to fruit from healthy trees. Moreover, cover-colour, equatorial-diameter, and weight were reduced, and fruit were softer, failing to meet the criteria to be properly commercialized and exported to demanding markets.
RESUMO
Silverleaf is caused by the fungus Chondrostereum purpureum, which produces wood necrosis and foliar silvering in woody plants. Field observations and studies in apple have shown the reversion of foliar symptoms. Because plants were clones and received identical agronomical management, it was hypothesized that reversion is driven by endophytic microbiota. Thus, the objectives of this study were to compare healthy, diseased, and reverted plants with respect to their physiology, endophytic microbial communities, antagonistic ability of their endophytes against C. purpureum, and defense genes expression. Water potential, stomatal conductance, chlorophyll content, and fluorescence were measured. Endophytic bacterial and fungal DNA were analyzed by denaturing gradient gel electrophoresis, and community richness and similarity were calculated. Wood cores were collected and bacterial and fungal endophytes were isolated and confronted with C. purpureum-virulent strains in dual-culture assays. Defense genes expression was measured by quantitative PCR. Results indicated that there were no differences in physiological parameters between healthy and reverted plants, except for fluorescence, and both type of plants differed from diseased ones. Bacterial and fungal community richness was similar in healthy and reverted plants and higher than in diseased ones. Endophytes from reverted and healthy plants showed high antagonism to C. purpureum. Furthermore, nonexpressor of pathogenesis-related gene 1 expression was upregulated in reverted plants, whereas phenylalanine ammonia lyase and polygalacturonase-inhibiting protein genes showed higher values in diseased plants. Overall, physiological, molecular, and microbial characteristics were similar between healthy and reverted plants, and both differed from diseased ones. Therefore, reversion of symptoms is associated with changes in the endophytic microbiota, which seems to be a promising source of biological control agents against C. purpureum.
Assuntos
Malus , Doenças das PlantasRESUMO
Silverleaf caused by the basidiomycete Chondrostereum purpureum affects numerous woody species, including fruit tree crops like apple, resulting in wood necrosis and foliar silvering. There are no curative alternatives for this disease, and its management is by prevention methods. Therefore, the aim of this study was to develop a rapid diagnostic tool for the detection and identification of C. purpureum directly from woody tissues to help distinguish the pathogen from other basidiomycetes that are commonly found on apple. The silverleaf pathogen was isolated from different hosts and locations, and Koch's postulates were performed by inoculating the isolates on apple cuttings and measuring internal necrosis. A previously described APN 1 pair of primers specificity was also tested against 25 C. purpureum isolates in this study, using other wood rotting species as negative controls. Seven virulent isolates were inoculated on apple cuttings, and DNA was extracted from the cuttings' sawdust and amplified using APN 1, after 22 days of incubation. To prove the efficiency of the method in the field, DNA from healthy nursery plants inoculated with two virulent isolates, and naturally infected plants showing different levels of foliar symptoms, were tested. Presence of the fungus was verified by reisolation on APDA in all assays. Koch's postulates indicated that all C. purpureum isolates were pathogenic, showing different virulence levels, and APN 1 primers were able to discriminate them from other basidiomycetes. The method was also able to detect C. purpureum from artificially inoculated plants as well as naturally infected ones, demonstrating that the protocol may become a rapid minimally destructive diagnostic tool to detect the pathogen without the need to isolate it from tissues, and thus taking measures to prevent its dissemination.