Advances in Environmental Technology and Biotechnology

Research on the biology of the brassicae isolated from mustard

CRISTINEL RELU ZALĂ, STELICA CRISTEA, LIVIU GRUIA, MALI-SANDA MANOLE Plant Science Department University of Agronomic Sciences and Veterinary Medicine, Bucharest 59 Marasti Blvd District 1, 011464, Bucharest ROMANIA [email protected]

Abstract: Alternaria brassicae is a plant pathogen able to infect most species including mustard. In Romania Alternaria black spot of mustard, incited by Alternaria brassicae. Alternaria brassicae fungus can be manifesting on every plant organ in every stage of plant development. The abiotic factors play an important role during the fungus development and pathogenicity. The study must be done in order to establish its needs regarding certain abiotic factors such as: temperature, humidity, and light. Different humidity values were created in dessicators, from 15% up to 100% R. H., using super concentrated solutions of some salts.The study is necessary in order to get to know the moment when the first infections occur. Mustard plants are attacked by conidia of A. brassicae (Berk.) Sacc., and A. brassicae is the dominant invasive species. It causes damping off if infection occurs in younger plants and severe leaf spot symptoms on infections of older plants. Infected seeds with spores on the seed coat or mycelium under the seed coat are the main means of distribution for these pathogens. The fungus can overwinter on susceptible weeds or crop debris and on seed plants, as well as on stecklings. Latent infections can occur and result in post-harvest diseases or damping-off in case of infected seed.

Key-Words: Alternaria brassicae, mustard, biology, conidia, symptoms, temperature, humidity, germination.

1 Introduction the photosynthetic area and accelerate senescence in the plant [4, 6, 8]. Infected mustard leaves develop Mustard (Sinapis alba L. syn. Brassica alba L.) brownish spots covered with black bloom of spores. plants in Romania are severely affected by the Latent infections can occur and result in post- Alternaria brassicae (Berk.) Sacc. [10]. Alternaria harvest diseases or damping-off in case of infected brassicae fungus can be manifesting on every plant seed [11]. This pathogen are responsible for major organ in every stage of plant development. The seed yield losses in the mustard because infected abiotic factors play an important role during the silique remain small, break open, and the seeds fungus development and pathogenicity. Therefore, a shook, and this is the most important component of certain biological study is necessary. The study their economic impact. A. brassicae belongs to: concerns the research of the Alternaria brassicae Kingdom Fungi, Phylum ; Subdivision pathogen fungus biology. The study must be done in Pezizomycotina; Class ; Order order to establish its needs regarding certain abiotic ; Family ; Subfamily factors such as: temperature, humidity, and light. mitosporic Pleosporaceae; Genus Alternaria [7]. The study is necessary in order to know the moment Alternaria fungi proliferation is vegetative in when the first infections occur [9]. Due to weather character, and takes place by means of conidial conditions conducive to infection, the disease spores, airborne and found in the soil and water, as constitutes a serious problem in mustard plant well as indoors and on objects. Sexual production in long-lasting high humidity or recombination (teleomorphy) occurs very rarely [8]. intensive rains regions from Romania. A. brassicae Development of the pathogen infection structures cause severe economic losses. Seed infection causes depends primarily on the incubation temperature reduced germination and seedling vigour, in [1], and relative air humidity. While spores of A. addition to pre- and post-emergence damping-off, brassicae germinate in a rather broad temperature and affects the sale and use of infected/infested range, germination effectiveness is correlated with seed. Lesions on leaves, stems, and siliques reduce the temperature [3]. High air humidity (95-100%

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RH) lasting at least 9-18 h is a crucial requirement each of them was placed in thermostates at displayed by A. brassicae during plant infection [5]. temperatures of 2-42°C. At a 3 days interval, the constant colonies diameter growth was registered. Also, fructifications growth was registered. The 2 Materials and methods observations lasted for 15 days. The colonies growth In laboratory conditions, phytopathogenic fungus and fructification of Alternaria brassicae fungus are Alternaria brassicae was grown on artificial agar influenced by temperature (Table 1). media with PGA (potato-glucose agar). The minimum temperature for the colony formation The sick biological material of mustard, which was was 2°C; they occurred under the form of a lax formed from leaves (Fig. 1) with specific disease mycelium, of light-grey colour, with a grey back. symptoms was put in a wet room, where, the Fructifications were absent. The aspect was the mycelium formation was observed after 3 days. same at 4°C and 6°C. The 8°C temperature Placed on PGA culture medium again, in Petri determined a better colony development, so that the recipients, colonies were formed; after 6 days, they mycelium was compact, with silky aspect, of grey purified the Alternaria brassicae species. The colour, with alight-grey back. The conidions abiotic factor was established in laboratory presence was registered. They were rare on the conditions. They influenced the Alternaria brassi- mycelium’s surface. Temperatures between 14 and cae fungus development, after Tuite’s method [2]. 26°C favored the emergence of a good vegetative mass. Optimal hyphae growth temperature for A. brassicae is 28-32ºC when 70 mm colony diameter was registered, with silky aspect, dense, grey in colour, with the light-grey back. Fructification was very good. Dark septa divide the branched or unbranched conidiophores, carrying the conidial chains. Growing hyphae develop light-brown or dark-tawny, conidions (after 8 days) - single or forming long chains with longitudinal and transverse septa (Fig. 2).

Fig. 1, Alternaria brassicae - Characteristic concentric lesions on the upper leaf side.

The relative humidites were controlled using the following: LiCl 150g (15.0% R. H.), CaCl2, 6 H2O (35.0% R.H.), Ca(NO3), 4 H2O (55.0% R. H.), Mg(C2H3O2)2 x H2O (65.0%), NaCl 304g (76.0%), NaCl 262g (80.0%), saturated aqueous solutions of Na2CO3 (92.0%), glycerol solution in distilled water (respectively 10%, 20%, and 40% per weight of glycerol to give 98%, 95% and 85% R.H.) Fig. 2, Alternaria brassicae - Conidia visible under light microscope [40x]. and H2O dist (100.0% R. H.). The dessicators were placed at 20°C with 16-24 hours photoperiod. The maximum value of temperature can be considered to be at 42°C. The formed colonies have a weak aspect, and fructifications were not even 3 Results and discussions formed. In order to analyse the influence of As observed on PGA culture medium, the temperature upon the conidia germination, in Petri Alternaria brasicae fungus develop fast-growing recipients and water-agar environment, the fungus thick colonies which are white-gray, with silky conidia were arranged; they were placed in aspect and dark-grey reverse. Alternaria brassicae thermostates at temperatures between 2°C and 42°C, fungus was placed on PGA culture medium again, and kept for 24 hours in these conditions. in Petri recipients of 8 cm in diameter, and then

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Table 1 At a 2 hours interval, germination was examined in Influence of temperature upon colony development 100 conidia for each variant. of the Alternaria brassicae The minimum threshold for conidia germination is possible at 4°C, and 16 hours are necessary (Fig. 3). Day 3 6 9 12 15 Observations The optimum temperature can be considered the 28- 32°C temperature interval, when 79-83% conidia tºC Diameter of colony after 15 days are germinated. (mm) The maximum temperature was 40°C and letal temperature was identified at 42°C when conidia did 2 0 0 2 4 10 Vm± Fr0 not germinate even when the Petri recipient was further set to an optimum temperature of 32°C. 4 0 1 10 13 14 Vm+ Fr0

6 0 1 10 13 14 Vm± Fr0 Germination (%) 90 8 0 2 10 16 15 Vm+ Fr± 80

10 0 4 14 27 24 Vm+ Fr+ 70 60 12 0 13 24 33 30 Vm+ Fr+ 50 14 2 12 25 30 33 Vm ++ Fr+ 40 16 2 12 25 32 38 Vm ++ Fr+ 30 18 2 14 25 36 38 Vm ++ Fr+ 20 10 20 2 15 26 37 40 Vm ++ Fr+ 0 2 6 22 2 17 28 37 40 Vm++ Fr+ 10 14 18 22 26 30 34 38 42 Temperature (°C) 24 2 18 30 38 40 Vm++ Fr+ Fig. 3, Germination conidia of Alternaria brassicae according to temperature. 26 2 22 34 38 40 Vm++ Fr+

28 3 30 65 68 70 Vm+++ Fr+++ We tested 11 relative humidities: (15%, 35.0%, 55.0%, 65.0%, 76.0%, 80.0%, 85.0%, 92.0%, 30 3 35 65 68 70 Vm+++ Fr+++ 95.0%, 98.0% and 100.0% R.H.). The Petri dish with PGA medium, in which the fungus were 32 3 38 65 68 70 Vm+++ Fr+++ placed, was introduced in dessicators, and kept for 15 days. The incubation Alternaria brassicae shows 34 3 40 65 66 68 Vm+++ Fr++ that infection develops dependently of the humidity (Table 2). At values of 15% R.H., the colonies were 36 2 40 65 58 60 Vm+++ Fr++ not formed. The development of the fungus is possible when the humidity values are over 36.8% 38 2 30 34 38 40 Vm++ Fr+ R.H.. At values of 55-76% R.H., the formed colonies had a thick-dark aspect, of grey colour, and 40 0 15 17 17 18 Vm+ Fr+ fructifications did not form. From values equal or superior to 76% R.H. the formation of conidia was 42 0 0 0 0 0 Stop the growth noted on the colony surface. With a higher relative Legend: Vm± = very weak vegetative mass; Vm+ = atmospheric humidity, the colony development is weak vegetative mass; Vm++ = good vegetative very good, and the vegetative mass is extremely mass; Vm+++ = very good vegetative mass; 0 = dense, grey in colour. Also, fructifications are fungus did not fructify; Fr ± =very weak abundant. fructification; Fr + = weak fructification; Fr.++ = On permanent or alternative light/darkness (12/12 good fructification; Fr+++ = abundent fructification. and 8/16), the colony vegetative mass was rich, had

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a silky mycelium, of grey colour, and sporulation Light is an important factor under natural conditions was abundant. of infection.

Table 2 Influence of relative atmospheric humidity upon References: colony development of the Alternaria brassicae [1] Bassey E.O., Gabrielson R.L. The effects of humidity, seed infection level, temperature and R.H. Diameter of Observations nutrient stress on seedling disease caused by . Seed (%) colony (mm) Science and Technology, Vol 11, 1983, pp. 403-410. after 15 days [2] Constantinescu O., Metode şi tehnici în micologie. Ed. Ceres, Bucureşti, 1974. 15 0 Colonies are [3] Degenhardt K. J., Petrie G. A., Morrall R. A. A., Effects of temperature on spore germination not formed and infection of by Alternaria brassicae, A. brassicicola, and A. raphani. 35 20 Weak growth Canadian Journal of Plant Pathology, Vol. 4, 1982, pp. 115-118. 55 32 Vm± Fr. 0 [4] Humpherson-Jones F.M., Maude R.B., Studies on the epidemiology of Alternaria brassicicola 65 37 Vm± Fr. 0 in Brassica oleracea seed production crops. Annals of Applied Biology, Vol. 100, 1982, pp. 76 65 Vm++ Fr. + 61-71. [5] Humpherson-Jones F.M., Phelps K., Climatic 80 70 Vm+++ Fr. ++ factors influencing spore production in Alternaria brassicae and Alternaria 85 70 Vm+++ Fr. +++ brassicicola. Annals of Applied Biology, Vol. 114, 1989, pp. 449-458. 92 70 Vm+++ Fr. +++ [6] Humpherson-Jones F.M., Survival of Alternaria brassicae and Alternaria 95 70 Vm+++ Fr. +++ brassicicola on crop debris of oilseed rape and cabbage. Annals of Applied Biology, Vol. 115, 98 70 Vm+++ Fr. +++ 1989, pp. 45-50. [7] Kirk P.M., Cannon P.F., Minter D.W., Stalpers 100 70 Vm+++ Fr. +++ J.A., Dictionary of the Fungi. 10th ed. Wallingford, 2008. Permanent darkness throughout the whole [8] Nowicki M., Nowakowska M., Niezgoda A., experiment led to a very weak vegetative mass, and Elżbieta U. Kozik E. U., Alternaria Black Spot conidia appeared very rarely on the mycelium of Crucifers: Symptoms, Importance of surface. Disease, and Perspectives of Resistance Breeding. Vegetable Crops Research Bulletin, 4 Conclusion Vol. 76, 2012, pp. 5-19. The abiotic factors (temperature, relative [9] Radu E., Stelica Cristea, C. R. Zală, Research atmospheric humidity and light) are important on the biological features Alternaria brassicae factors in the Alternaria brassicae fungus evolution. pathogen isolated on rape. Scientific Papers, Optimum temperature for hyphae growth and USAMV Bucharest, Series A, Vol. LIV, 2011, conidia germinations is between 28-32°C, when 79- pp.350-355. 83% of conidia are germinated. [10] Saccardo P.A., Alternaria brassicae (Berk.) Alternaria brassicae as many other fungal Sacc. Michelia Vol. 2, No. 6, 1880, pp. 129. pathogens depend on the presence of free moisture [11] Thomma B. P. H. J., Alternaria spp.: from on the host or of high relative humidity (superior general saprophyte to specific parasite. 80.0% R.H.) durind the growing season for the Molecular Plant Pathology, Vol. 4, 2003, pp. conidia germination and such for infection and 225–236. development in the crop.

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