A PCR-Based Method for the Rapid Detection ofChondrostereum purpureumin Apple

Abstract

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.

Silverleaf (Chondrostereum purpureum) Effects on Japanese Plum (Prunus salicina)

“…Fungal trunk diseases have increased in recent years, in fruit, ornamental, forest, and woody species, worldwide [2]. Some of the reasons of this increase in trunk diseases in fruit crops are changes in climate, the incorporation of intensive plant production management measures such as high density plantations, the use of dwarfing rootstocks, and severe pruning, in order to accelerate plant and fruit production, which stress the plants and alter their behaviour [2][3][4]. Fungal trunk diseases are one of the main pathological problems for Prunus, which are affected by several fungal taxonomic groups [5].…”

“…While this fungal species has attracted considerable interest in several countries as a biological agent for woody weed control in coniferous forest plantations [13][14][15][16][17], it can cause severe and destructive disease in woody plants, including Plants 2021, 10, 2777 2 of 14 ornamental, wild, forest, and fruit crop species in some areas of the world [4,[17][18][19][20][21][22][23]. More than 230 species are recorded as hosts of C. purpureum [15,18,19,24,25], i.e., the Rosaceae family [3,4,18,19,[26][27][28] and genus Prunus, one of the most susceptible to this pathogen [18], such as P. armeniaca [29], P. avium [30], P. persica 49 [31], P. serotina [19], P. domestica [32], and P. salicina [13].…”

“…Silverleaf is an important disease in countries such as New Zealand, the United States (Washington and Oregon) [32], Chile [3], Australia [36], France [18], Poland [37], Latvia [38], Norway [39], and other European countries [32]. It can produce severe losses in orchards, and it is the main cause of mortality in peach and nectarine in New Zealand, with losses of 8% per year [40].…”

Silverleaf (<em>Chondrostereum purpureum</em>) Effects on Japanese Plum (<em>Prunus salicina</em>)

“…Pure cultures were incubated on PDA at 25°C for 7 days. Total nucleic acids were isolated from fresh mycelium using the CTAB method, and genomic DNA was amplified using APN 1 C. purpureum specific primers described by Becker et al (1999), following the protocols described by Grinbergs et al (2020). The PCR products (20 μl) were analyzed on 1.5% agarose gels, using a 1 kb DNA ladder (Maestrogen Inc., Xiangshan Dist., Hsinchu, Taiwan) as molecular size standard.…”

“…Fungal trunk diseases have been increasing in recent years, in fruit, ornamental, forest and woody species, worldwide. Some of the reasons of this raise in fruit crops are changes in climate, the incorporation of intensive plant production management measures like high density plantations, the use of dwarfing rootstocks and severe pruning, in order to accelerate plants and fruit production, which stress the plants and alter their behavior (Gramaje et al, 2016;Grinbergs et al, 2020, France et al, 2017. They are one of the main pathological problems for Prunus, which are affected by several fungal taxonomic groups (Gramaje et al, 2012).…”