STUDIES ON EARLY AND RELIABLE FIELD DETECTION OF Marssonina coronaria IN APPLE
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Date
2020-03
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NAUNI,UHF
Abstract
ABSTRACT
The present investigations entitled “Studies on early and reliable field detection of Marssonina coronaria
in apple” was undertaken to conduct the disease survey, study the overwintering structure development, molecular
identification, primer designing and standardization of LAMP and field detection of Marssonina coronaria using
LAMP. The disease survey was conducted in ten different areas of Himachal Pradesh. The survey report showed
maximum disease incidence (100%) in Seobag (Kullu) and minimum (72%) in Sangla (Kinnaur), where as, disease
severity was found maximum (68.2%) in Patlikuhal, Seobag (Kullu) and minimum (23.2 %) in Kumarsain, Narkanda
(Shimla). Leaf samples with Marssonina blotch symptoms were collected for isolation of pathogen. The five cultures
of Marssonina species were isolated and they were coded as Mashobra 1, Rohru 1, Rohru 2, Jubbal 1 and Jubbal 2.
These five isolates were having light brown to black colony colour without mycelium and condia of all isolates were
hyaline. Molecular identification of five different cultures was done by ITS gene sequencing and they were confirmed
as Marssonina coronaria. In the studies of overwintering structure development, acervuli were seen rupturing through
host leaf epidermis (asexual fruiting body) during December, 2018 to January, 2019. Acervuli were
pseudoparanchymatous, depressed and saucer shaped, developed beneath the host surface with 156-175 μm in
diameter and 69-71 μm in height. The initials of apothecium fruiting body were visible in the last week of January,
2019. The first apothecium with asci and paraphyses was observed in 1st week of March, 2019. However, fully
developed apothecium with asci and ascospores was visible after 4th week of March and 1st week of April, 2019.
Apothecia were fleshy, cup shaped 121-216 μm in diameter and 108-187 μm in height; asci broad-clavate to oblong,
45-74 × 12-19 μm, 8-spored; ascospores oblong elliptical, usually 1-septate, straight or somewhat curved, slightly
constricted at the septum or not so, acute or rounded at the ends, hyaline, 12-24 × 3-6 μm; paraphyses filiform, 1-2
septate, almost the same height as the asci, slightly broadened at the apex. The ascospores trapped in spore traps in
apple orchard were observed during 3rd and 4th week of April, 2019. Hollow apothecia were observed during 1st week
of May, 2019 and onwards. LAMP primers were designed using online software Primer Explore V4. The primers
were designed using the sequences obtained from DNA of Marssonina coronaria under study. The LAMP reaction
was standardized and the temperature of 64°C and 65°C were found best for detection of pathogen. Field detections
were done using standardized LAMP conditions in water bath. LAMP is easy to perform once the appropriate primers
are prepared, requiring only four primers, Bst reaction buffer, dNTPs, a DNA polymerase and a regular laboratory
water bath or heat block for reaction. Therefore, LAMP can be used for field detection of Marssonina coronaria
without any sophisticated equipments.