Simple diagnosis using ethanol immersion of strawberry plants with latent infection by Colletotrichum acutatum, Dendrophoma obscurans, and Fusarium oxysporum f. sp. fragariae

Abstract

Simple diagnosis by ethanol immersion (SDEI) to detect Glomerella cingulata was used to detect three other fungi that also cause latent infection of strawberry plants. Signs on strawberry leaves with asymptomatic latent infection by Colletotrichum acutatum became visible using SDEI. Salmon-pink conidial masses were produced in the acervuli on the treated leaves 5 days after incubation at 28°C. In the case of Dendrophoma obscurans, pycnidia with amber conidial masses formed 5 days after incubation at 28°C. The pycnidia were observed mainly on the ribs, and conidial masses exuded from the ostiole. These macroscopic conidial masses were similar to those of G. cingulata and C. acutatum. When water was dripped onto a lesion caused by D. obscurans, the pycnidia exuded white filamentous conidial masses, making the distinction of D. obscurans from G. cingulata or C. acutatum. On petioles with latent infection by Fusarium oxysporum f. sp. fragariae, white aerial hyphae grew out from the vascular tissues on the cut surface 3 days after incubation at 28°C and were easily observed by eye or with a loupe. Thus, SDEI was also useful for diagnosing latent infection of strawberry plants by C. acutatum, D. obscurans, and F. oxysporum f. sp. fragariae.

Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants

“…The number of samples per nursery ranged from 6 to 70 (average 42 samples/nursery) ( Table 2 ). Using our nested PCR assay, the detection rates ranged from 0% to 100% (average 20%); using the SDEI method [ 30 ], the detection rates ranged from 5% to 90% (average 45%) ( Table 2 ). For the samples from 16 out of 18 nurseries, the detection rates from the nested PCR assay were lower than those from the SDEI method.…”

“…From 2019 to 2020, 747 asymptomatic leaf samples (747 mother plants) collected from 18 strawberry nurseries in Hsinchu City, Miaoli County, Taichung City, and Nantou County were tested using the nested PCR and simple diagnosis by ethanol immersion (SDEI) methods [ 30 ] ( Table 2 ). In these nurseries, the mother plants were reproduced from field plants by the farmers themselves.…”

“…Culture-based methods are simple and convenient but a long time is required for sporulation; in addition, a great deal of experience is required to correctly differentiate Colletotrichum spp. from other fast-growing fungi with similar morphology [ 29 , 30 ]. Faster and more accurate PCR-based detection methods have also been developed, using specific primers targeting the internal transcribed spacer (ITS) region of ribosomal DNA [ 25 , 27 , 28 , 32 ], β-tubulin ( TUB2 ) gene [ 21 ], or mating type gene MAT1-2 [ 33 ].…”

“…Several methods for diagnosing latent infection or distinguishing Colletotrichum spp. infecting strawberry have been developed [21,[23][24][25][26][27][28][29][30][31][32][33]. For instance, culture-based methods involve incubation of strawberry tissues after surface sterilization with ethanol or killing with paraquat or freezing [24,[29][30][31].…”

“…infecting strawberry have been developed [21,[23][24][25][26][27][28][29][30][31][32][33]. For instance, culture-based methods involve incubation of strawberry tissues after surface sterilization with ethanol or killing with paraquat or freezing [24,[29][30][31]. These treatments promote the necrotrophic growth of the latently infected pathogens, resulting in the formation of yellow to orange conidiomata after 1-2 weeks of incubation.…”