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Available online at http://journals.usamvcluj.ro/index.php/promediu ProEnvironment

ProEnvironment 6 (2013) 59 - 63

Original Article

Studies Regarding the Morphological Identification of Fusarium sambucinum Fuckel Isolated from Potato Tubers

BORCA Ioana Daniela*, Carmen Emilia PUIA

University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Horticulture, Mănăştur no. 3-5, 400372, Cluj - Napoca, Romania

Received 13 January 2013; received and revised form 8 February 2 013; accepted 1 March 2013 Available online 31 March 2013

Abstract

Fusarium sambucinum Fuckel - teleomorph pulicaris (Fr.) Sacc. - is a common pathogen causing dry rot of stored tubers, in temperate areas. Fusarium dry rot is one of the most important diseases of potato (Solanum tuberosum L.), affecting the tubers in storage and the seed pieces after planting. To establish strategies for the control of this disease it should be made primarily a correct diagnosis and identification of the pathogen. The purpose of this study is to identify based on morphological characters the Fusarium sambucinum Fuckel isolated from potato tubers. The characters followed are: the colony growth diameters, the culture pigmentation, the shape and the size of the macroconidia, the presence and the absence of the microconidia and of the chlamydospores. The isolates were obtained in two ways: directly from the tuber tissue and from the diseased sprout formed after the tuber was maintain in the damp chamber. In this study, the length and the width of 100 conidia from each Fusarium sambucinum Fuckel isolate were measured, having three isolates obtained directly from the tuber tissue and three isolates from spores formed on the surface of the tuber after keeping them in damp chamber conditions.

Keywords: Fusarium, isolates, conidia, potato.

1. Introduction The pathogen transmission is correlated with it ability to sporulate underground on the seed tubers It was reported that 60% of qualitative tubers or on the stem bases, Fusarium sambucinum Fuckel can be affected of Fusarium dry rot during storage sporulates on the stem [1, 5]. and the annual crop losses are estimated to average The first symptoms of Fusarium dry rot are from 6 to 25% [4]. manifested by the appearance of dark depressions on In storage the dry rot develops most rapidly at the surface of the tubers. In large lesions, the skin a relative humidity of 50-80% and at a temperature becomes wrinkled in concentric rings as the of 15 - 28oC. Lower value of humidity and underlying dead tissue desiccates [6]. temperature retard the infection and the disease The internal symptoms are characterized by development [7]. The transmission of Fusarium necrotic areas. This necrotic tissue is usually dry sambucinum Fuckel to the progeny tubers is greater (hence the name ”dry rot”) and may develop at an from the highly contaminated seed then from the injury such as a cut or bruise [6]. rotting [1, 4, 5]. The pathogen enters the tuber causing the rot, often rotting out the centre. The rotted cavities are often lined with mycelia and spores of different * Corresponding author. Tel.: 0040264401664; Fax: 0040264 592 055 colours from yellow, to white or even pink [2]. e-mail: [email protected]

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The lesions caused by the infection with In both of the cases it was used the single spore Fusarium sambucinum Fuckel tend to be dark technique to obtain a pure culture of Fusarium brown to black and progresses inside the tuber in a sambucinum Fuckel, from tissue samples and very irregular manner having the aspect of tunnels diseased sprout samples. [1]. Fusarium sambucinum Fuckel - sexual stage The isolate of Fusarium sambucinum Fuckel Gibberella pulicaris (Fr.) Sacc. – belongs to the were grown on PDA using 85 mm Petri plates. The phylum Ascomycotina, the class , Petri plates were sealed with plastic foil to avoid the the order , the family and contamination and they were incubated at 25oC for 4 the genus Gibberella [8]. days. On the fourth day were made the observation The identification of Fusarium sambucinum on the growth and the development of Fusarium Fuckel isolated from potato tubers is performed sambucinum Fuckel colonies by measuring their after obtaining a pure culture using the single spore diameters. After a period of 10 - 14 days with daily technique and were made observations regarding the exposure to light the observations were made on the colony growth diameters on agar media (PDA) after aerial mycelia characteristics and on the colonies an incubation in the dark for 4 days (at 25oC), the reverse (the pigmentation). culture pigmentation on agar media (PDA) after an For the microscopically identification of the incubation for 10 - 14 days with a daily exposure to pathogen the observations were made on the shape light and the microscopic morphology including and the size of the spores. shape and size of the macroconidia, the presence or We have measured the spores with the Motic the absence of the microconidia and of the Images 2000 program and were measured the length chlamydospores [3]. and the width of 100 conidia from each Fusarium sambucinum Fuckel isolate. 2.Material and Method 3.Results and Discussions In this study, for the identification of Fusarium sambucinum Fuckel isolated from potato For the macroscopically phase of the tubers we followed the steps presented in fig. 1. pathogen identification we’ve made observations on The pathogen isolation was made from both the growth and development of the colonies by infected tuber tissue (in vivo) and diseased sprouts measuring the diameter of growth, but also on the (in vitro) form in the damp chamber conditions, on colonies aspect by describing the aerial mycelia and Potato Dextrose Agar (PDA). the colonies reverse (the pigmentation). To isolate Fusarium sambucinum Fuckel from In table 1 are presented the colony diameters of the the spores, the analyzed potato tuber was held in a isolates, developed on the media used (PDA) and damp chamber to encourage a better sporulation of measured after an incubation of 4 days in the dark the . (at 25oC).

Table 1. The diameters of Fusarium sambucinum Fuckel colonies formed on the culture media (cm) Isolate Diameters (cm) in vivo in vitro F1 4.4 5.9 F2 5.8 5.7 F3 5.4 5.9

The observations regarding the aspect of the The spores produced by the fungus culture on colonies (fig. 2) were made after a period of 10 - 14 the surface of the agar media, vary in abundence. days with daily exposure to light and are presented They are usually abundant in sporodochia in table 2. which usually are orange and form in the center of In fig. 3 it can be seen that the macroconidia the culture. have a falcate shape, are slender, comparatively The length of the macroconidia (table 3) short and usually rather uniform in size. isolated from the tuber tissue took values between The apical cell is pointed and the basal cell is 33.5 (F1 isolate) and 32.1 (F3 isolate). foot shaped. The number of septa is 3 usually 6 The width (table 3) had the lowest value in the septate. The microconidia are very rare, but found in case of F1 isolate (4.2 μm) and the highest values in the aerial mycelia when are present, they have an the case of F3 isolate (5.0 μm). The length of the oval shape with 0 to 1 septate. macroconidia isolated from the tuber diseased

60 BORCA Ioana Daniela et al./ProEnvironment 6(2013) 59 - 63

sprouts and grown on PDA media recorded the being 32.3 μm and the lowest value on F1 isolate biggest value in the case of F3 isolate, the average were the average was 31.1 μm.

DISEASED PLANT

Isolation

RECOVERY OF FUSARIUM

Do you need to NO Fusarium sp. progress further? YES SUBCULTURING ONTO PDA

PURIFICATION

IDENTIFICATION PROCESS

COLONY MORPHOLOGY THE MORPHOLOGICAL CHARACTERS OF THE SPORES

GROWTH RATES MACROCONIDIA  AERIAL MYCELIA shape CHARACTERISTICS  size  formation COLONY REVERSE (PIGMENTATION) MICROCONIDIA  shape  size  abundance

INFORMATION GATHERING (morphological, physiological, host, TAXON geographic) INFORMATION

1. The Figureflow chart 1. The of flow the chart protocol of the protocoused forle usedthe identificationfor the identification of the of theFusariumFusariumsp. sp.

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Table 2. The aspect of Fusarium sambucinum Fuckel on culture media (PDA) Isolate Aerial mycelia traits Colony reverse (pigmentation) in vivo in vitro in vivo in vitro F1 white to pink, white to gray, bloody red Yellow floccose, abundant floccose, abundant (ruby red with brown dots) (orange) F2 pink, floccose abundant white-pink, floccose, pink bloody red abundant, in center (ruby red) (ruby red with brown dots) sporodochia F3 white to pink white to pink, bloody red dark pink floccose, abundant, floccose, abundant, (ruby red with brown dots) (ruby red with brown dots) in center sporodochia in center sporodochia

Figure 2. The colony aspect (front/back) of Fusarium sambucinum Fuckel colonies on culture media (original)

Figure 3. The microscopic view of Fusarium sambucinum Fuckel macro- and microconidia, isolated from potato tubers

The width of the same macroconida isolated dimension was obtained in the case of F2.isolate (4.3 from the tuber diseased sprouts recorded the highest μm). In none of the isolates have been reported the value in F1 isolate (5.0 μm) and the lowest average formation of chlamydospores.

Table 3. The length/width average of the Fusarium sambucinum Fuckel macroconidia (μm) Conidia size (μm) Isolate in vivo in vitro Length Width Length Width F1 33.5 4.2 31.1 5.0 F2 32.4 4.3 31.5 4.3 F3 32.1 5.0 32.3 4.4

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4.Conclusions [2] Boyd A.E.W., 1972, Potato storage diseases, Rev. Plant Pathol. 51, 279 - 321 The colonies grown on PDA media had the diameters between Fusarium sambucinum Fuckel [3] Burgess L.W., C.M. Liddell, 1983, Laboratory manual for specific limits. Fusarium research, Fusarium Research Laboratory, Department of Plant Pathology and Agricultural Entomology, The aspect of the colonies grown on culture The University of Sydney, Australia media is typical for the identified species (Fusarium sambucinum Fuckel). [4] Carnegie S.F., A.M. Cameron, P. Haddon, 2001, The The microscopic observations and the effect of date of haulm destruction and harvest on the measurements indicate that the analyzed development of dry rot caused by Fusarium solani var. macroconidia have a falcate shape with a „pointed” coeruleum on potato tubers, Ann. Appl. Biol. 139, 209 - 216 apical cell and a ”foot shaped” basal cell. The recorded dimensions of the macroconidia [5] Choiseul J.W., L. Allen, S. . Carnegie, 2001, The role are between Fusarium sambucinum Fuckel specific of stem inoculum in the transmission of Fusarium limits and the microconidia are very rare, a typical sulphureum to potato tubers, Potato Res. 44, 165 - 172 character of the identified specie. [6] Secor G.A., B. Salas, 2001, Fusarium dry rot and Fusarium wilt in: Compendium of Potato Disease, 2nd ed., eds. The American Phytopathological Society, St. Paul References [7] Wharton P.S., W.K. Kirk, D. Berry, P Tumbalam, 2007, Seed treatment application-timing options for control of [1] Adams M.J., D.H. Lapwood, 1983, Transmission of fusarium decay and sprout rot of cut seedpieces, American Fusarium solani var. coeruleum and F. sulphureum from Journal of Potato Research, 237 - 244 seed potatoes to progeny tubers in the field, Ann. Appl. Biol. 103, 411 - 417 [8] *** www.catalogueoflife.org

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