2012 vol. 76, 5-19 DOI: 10.2478/v10032-012-0001-6 ______

ALTERNARIA BLACK SPOT OF CRUCIFERS: SYMPTOMS, IMPORTANCE OF DISEASE, AND PERSPECTIVES OF RESISTANCE BREEDING

Marcin NOWICKI, Marzena NOWAKOWSKA, Anna NIEZGODA, Elżbieta U. KOZIK Research Institute of Horticulture Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland Received: June 6, 2012; Accepted: July 16, 2012

Summary black spot of cruciferous vegetables, incited by different species of Alternaria, remains an increasing threat to crops throughout the world, including Poland. plants are attacked by conidia of A. brassicae (Berk.) Sacc., A. brassicicola (Schw.) Wiltsh., A. raphani Groves & Skolko, and A. alternata (Fr.) Kreissler. The have a wide spectrum of hosts, such as head , Chinese cabbage, cauliflower, , and other crucifers includ- ing cultivated and wild grown plants. Alternaria pathogens usually cause damp- ing-off of seedlings, spotting of leaves of , blackleg of heads of cabbag- es, and spotting of cauliflower curds and broccoli florets. In oilseed rape, A. brassicae is the dominant invasive species, while in the cruciferous vegetables, both species, A. brassicae, and A. brassicicola are encountered. Infected seeds with spores on the seed coat or mycelium under the seed coat are the main means of distribution for these pathogens. The can overwinter on susceptible weeds or crop debris and on seed plants, as well as on stecklings. Methods for disease prevention and control are based on combining agri- cultural management practices with chemical control. Using disease-free seeds or seeds treated with fungicides can greatly reduce disease incidence. After appearance of the first symptoms of disease, stringent fungicide spray program is an effective way to reduce losses. Many authors seem to agree, that the most economically feasible method of disease control is the development of resistant Brassicaceae crops varieties, as transgenic approach proved unsuccessful. Due to our increasing understanding of -host plant interactions, identifica- tion of resistance sources, and assessment of the resistance trait inheritance mode, breeding programs of Brassica crops for Alternaria resistance can be enhanced. This is of particular importance since recent years experience dynam- ic development of ecological and integrated plant production with an emphasis on plant biotic stress resistance. Highly resistant genetic resources have not been reported in Brassica cultivated species, although some varieties differ in their resistance/susceptibility level.

Corresponding author: e-mail: [email protected] © Copyright by InHort

6 VEGETABLE CROPS RESEARCH BULLETIN 76 ______Strong cross-incompatibility, polygenic background of the resistance (ad- ditive and dominant gene interactions), as well as the differences in ploidy be- tween the Brassica species of interest, render the transfer of Alternaria re- sistance from the wild species into the cultivated forms difficult. Additionally, it is often connected with employment of in vitro hybridization techniques, in- cluding somatic hybridization, and ovary rescue, or protoplast fusion. key words: Alternaria spp., cruciferous plants, resistance, testing methods

INTRODUCTION A. brassicicola and A. brassicae cause severe economic losses in sev- Cruciferous plants (Brassicaceae) eral different ways (Humpherson- worldwide are severely affected by the Jones & Maude 1982, Humpherson- Alternaria fungi. A. brassicae (Berk.) Jones 1989). Seed infection causes Sacc., A. brassicicola (Schw.) Wiltsh., reduced germination and seedling A. raphani Groves and Skolko, and A. vigour, in addition to pre- and post- alternata (Fr.) Kreissler belong to spe- emergence damping-off, and affects cies of major negative influence on the sale and use of infected/infested Brassicaceae plants. Although both, seed. Lesions on leaves, stems, and A. brassicicola and A. brassicae occur siliques reduce the photosynthetic on oleiferous and vegetable (olera- area and accelerate senescence in the ceous) , the former is the dom- plant. These pathogens are responsi- inant invasive species of the vegetable ble for major seed yield losses in the Brassicas, while the oleiferous cruci- oleraceous Brassicas and this is the fers are primary hosts for the latter most important component of their fungus (Maude & Humpherson-Jones economic impact. The unsightly cos- 1980, Humpherson-Jones 1989). metic blemishing or rotting of the Both most common Alternaria head or wrapper leaves in vegetable pathogens usually cause black spot Brassicas as a consequence of patho- disease, manifested by damping-off of gens’ toxins and disease causes seedlings, spotting of leaves of cab- downgrading and crop losses in both bages, blackleg of heads of cabbages fresh and stored produce. A. bras- (head cabbage and Chinese), and spot- sicicola often occurs in conjunction ting/browning of cauliflower curds with A. brassicae and some other and broccoli florets. Black spot is the pathogens of the Brassicaceae. This most common disease in the crucifers confounds precise estimates of losses plantations located in tropical and caused individually by this pathogen sub-tropical regions. Due to weather in the field. conditions conducive to infection (see In Europe, alone the seed losses below), however, the disease consti- due to both pathogens were estimated tutes a serious problem in crucifers at up to 86% in B. oleracea in several plant production in long-lasting high years (Maude & Hampherson-Jones humidity or intensive rains regions, 1980, Humpherson-Jones 1989). Un- including Poland. der Polish climate conditions, the disease takes particularly high toll on

M. NOWICKI et al. – ALTERNARIA BLACK SPOT … 7 ______the late and medium-late varieties of active ingredients (Amistar 250 SC, head-cabbage grown for sauerkraut Signum 33 WG, Zato 50 WG) and processing, as well as for storage. fungicides based on iprodione (Rovral Cabbage heads with characteristic FLO 255 SC) (Maude et al. 1984, symptoms of Alternaria black spot do Survilienė et al. 2010). This method, not store well, and their processing however, carries an economic disad- value is very low. This translates onto vantage and may prove ineffective significant economic impact of the under pathogen infection-conducive disease: In 2010, Polish cabbages and weather conditions, particularly other Brassicas production ranked 7th, among the seed crops. An alternative while cauliflower and broccoli ranked protection method to be employed is 8th globally (1141200 tonnes and use of antagonistic fungi; deployment 252325 tonnes, respectively) with net of and Epi- worth of production of Int.$171 M and coccum nigrum on the crucifers leaves Int.$60,5 M, respectively (FAOSTAT: reduced the infection level under con- http://faostat.fao.org). trolled conditions (Pace & Campbell Alternaria prevention and con- 1974). Field studies concerning the trol methods include combining the biological control efficacy are yet to proper agro-technique with chemical be carried out. protection. An essential disease pre- Pathogen profile and infection pro- vention method is production of gress healthy seeds, obtained from planta- The current classification of Al- tions with heavy fungicide protection. ternaria fungi is as follows: Kingdom In the 2-year lasting crucifers seed Fungi, Phylum ; Subdivi- production periods, good effects of sion Pezizomycotina; Class Dothide- protection against Alternaria infec- omycetes; Order ; Family tions during the 1st year of growth ; Subfamily mi-tosporic were expedited by fungicides contain- Pleosporaceae; Genus Al-ternaria ing iprodione as an active ingredient (http://www.uniprot.org/taxon- (Maude et al. 1984, Survilienė et al. omy/5598). There are 299 species listed 2010). In Poland, the only product in the genus (Kirk et al. 2008); most Al- containing it is Seed Protector T 75DS ternaria species are saprophytes that are WS (Zaprawa Nasienna). Since both commonly found in soil or on decaying pathogens survive on crop debris, plant tissues (Bart & Thomma 2003). seeds, and in association with weed Alternaria fungi proliferation is vege- hosts (Humpherson-Jones & Maude tative in character, and takes place by 1982, Humpherson-Jones 1989), crop means of conidial spores, airborne and debris management (for example found in the soil and water, as well as through crop rotation and deep tillage) indoors and on objects. Sexual re- and use of clean seed and proper weed combination (teleomorphy) occurs control should alleviate the disease. very rarely. After appearance of disease symp- Alternaria spp. may be grown on toms, one may achieve limitation of artificial agar media, with PDA (pota- the infection by repeated spray with to-dextrose agar) and V8 (V8 juive - fungicides containing strobilurines as agar) being the most popular in use.

8 VEGETABLE CROPS RESEARCH BULLETIN 76 ______As observed in the in vitro cultures, Chinese cabbage, cauliflower, brocco- the pathogens develop fast-growing li, and of other crucifers. Infected thick colonies which are usually cauliflower curds or broccoli florets green-black, or white-gray, with develop slight dents with brownish brown to black reverse. Dark septa spots covered with black bloom of divide the branched or unbranched spores. In these plants, infection usual- conidiophores, carrying the conidial ly remains on the surface and does not chains. Growing hyphae develop reach deep in the curd or the floret; light-brown or dark-tawny, club- however, symptomatic cauliflower or shaped spores - single or forming long broccoli florets lose their commercial chains with longitudinal and trans- value. In case of radish, turnip, or ruta- verse septa (Fig. 1A). baga, the disease affects the root thick- Primary source of pathogens are enings as well; the disease symptoms the infected seeds or non-decomposed manifest themselves as brown rots only plant debris in the top soil layers with during their storage. over-wintering hyphae or spores. An- Three ways of Alternaria infec- other important source of the patho- tion have been reported: Through gen are the Brassica weeds, which penetration, through - promote infestations as pathogen host ta, and through insects- or agrotech- plants (Humpherson-Jones 1989). nique-derived host plants wounding. During the vegetation period, the rain- Regardless of their means of entry, A. and wind-transported fungal conidial brassicicola and A. brassicae exhibit spores are also an important source of distinct differences in the host plant infection. A majority of conidia are tissue penetration. A. brassicae in- released during harvest and cleaning vades host plants solely through their the crops from the infected leaves; stomata, while for A. brassicicola, such released spores are then spread direct plant tissue penetration prevails approximately within 1,800 m over stomatal infections. Hyphae of (Humpherson-Jones & Maude 1982). both pathogens develop well on the Under Polish climate, highest Al- epidermis, directly beneath the leaf ternaria spore concentrations are de- waxes, and exhibit low cell penetra- tected in the air mainly in June and tion ratio. Upon successful pathogen July (Nowakowska et al. 2011). attack, dark-brown spots of different Alternaria black spot symptoms sizes (0.5 cm to several cm in diame- appear on all host plant parts and at ter) appear on the leaves; the spots of every developmental stage. A. bras- characteristic concentric circumfer- sicicola and A. brassicae cause the ences sometimes have a yellow chlo- damping off of the crucifers seedlings. rotic halo (Fig. 1B,C). Host plants' Elongated brownings develop on the reaction to the perceived infection is sub-cotyledonous part of the stem and manifested as almost immediate on the cotyledons, often leading to browning of cell walls, in particular in narrowing and breaking of the stems, the parastomatal cells. Under favora- and thus, to seedlings' decease. Most ble conditions, lesions become cov- often infected are the lower, older ered with brown-black downy-like leaves of head cabbage (Fig. 1B,C), bloom of sporulating hyphae. A. bras-

M. NOWICKI et al. – ALTERNARIA BLACK SPOT … 9 ______sicicola-derived spots are darker and and the infected plant tissue perishes less regular in shape compared with and crumbles, giving rise to dents and those of A. brassicae origin. As the hollows (Fig. 1C). disease progresses, the spots enlarge,

A B

C

Fig. 1. and cabbage damping off symptoms. (A) Pathogen my- celium as visible under light microscope [40x]. (B) Characteristic concentric le- sions on the abaxial leaf side. (C) Due to heavy infection, seedlings display black spot symptoms, followed by damping off.

Development of the pathogen’s spores of both Alternaria species infection structures and of the disease germinate in a rather broad tempera- symptoms on the oleraceous Brassicas ture range, germination effectiveness depends primarily on the incubation is correlated with the temperature (De- temperature (Bassey & Gabrielson genhardt et al. 1982). Optimal hyphae 1983), and relative air humidity. While growth temperature for A. brassicae is

10 VEGETABLE CROPS RESEARCH BULLETIN 76 ______18-24ºC, and for A. brassicicola 20- rotic lesions. Destruxin B was shown 30ºC. According to the in vitro studies, to be a major one responsible for in- sporulation of A. brassicae is tempera- ducing necrotic lesions on plant leaves ture-dependant: At 8-24ºC, fully- and eliciting the phytoalexins developed spores are detectable at 24 brassilexin and sinalbin A (Sodelade et or 14 h, respectively. Temperature al. 2012). Majority of researchers clas- spectrum of A. brassicicola sporulation sify destruxin B as an HST (Sodelade is broader (8-30ºC), with fully- et al. 2012), while others have demon- developed spores detectable after 43 or strated its unspecific character and 14 h, respectively. High air humidity questioned its role in the initial host (95-100% RH) lasting at least 9-18 h plant colonization (Buchwaldt & is a crucial requirement displayed by Green 1992, Parada et al. 2008). While both pathogen species during plant some isolates of A. brassicae produce infection (Humperson-Jones & Phelps destruxin B as their sole toxin, others 1989). Reports of massive infestations are capable of production of their de- under air temperatures of 20-27ºC and rivatives (homodestruxin B, constant plant moisture of at least 5 h, desmethyldestruxin B, and destruxin or RH exceeding 95% lasting at least B2; Parada et al. 2008). 12-20 h are commonplace (reviewed Depudecin, an eleven-carbon in Bart & Thomma 2003). linear polyketide and histone deacety- During plant infection, both Al- lase (HDAC) inhibitor made by ternaria fungi produce and exude A. brassicicola, proved a minor viru- phytotoxins that belong to HST clade lence factor. Depudecin-minus mutants (host-specific toxins or host-selective have a small (10%) but highly signifi- toxins; Parada et al. 2008, Wight et al. cant (p<0.01) reduction in lesion size 2009). These compounds play a major on cabbage, but not on Arabidopsis, role in the pathogenesis by determin- including the pad3 mutants, a suscep- ing the host plant spectrum, as well as tible control for Alternaria inoculation the isolates’ and pathogenic- (Zhou et al. 1999, Wight et al. 2009). ity levels (Nishimura & Kohmoto Likely reasons for only a minor role of 1983). To date, two HSTs have been this polyketide in A. brassicicola path- characterized in detail: AB toxin of ogenicity on cabbage and Arabidopsis A. brassicicola (Otani et al. 1998) and are: Depudecin concentrations or pene- ABR of A. brassicae (Parada et al. tration insufficient to effectively in- 2008). Both phytotoxins are proteins hibit host plant’s HDAC; HDAC in- with a suggested role in evoking dis- hibition playing major role in patho- ease symptoms on the infected crucif- genicity in grasses but not in other erous plants. plants (cabbage, Arabidopsis); redun- In addition to the above, dancy of HDAC inhibitors in A. brassicae produces a number of A. brassicicola, masking the depudecin phytotoxins (destruxin B and deriva- loss (Wight et al. 2009). tives, such as homodestruxin B, Alternaria spp. pathogens are also desmethyldestruxin B, and destruxin capable of production of other unspe- B2) responsible for typical black spot cific toxins. In all Alternaria - infected symptoms, such as necrotic and chlo- organs, alternariol and tenuazonic acid

M. NOWICKI et al. – ALTERNARIA BLACK SPOT … 11 ______have been detected. Alternariol- Sources of Alternaria spp. resistance induced cytotoxicity is mediated by Brassica crops endangered with activation of the mitochondrial path- Alternaria black spot necessitate way of . High concentrations complex projects on production of of tenuazonic acid inhibit protein syn- varieties with high levels of genetic thesis and, thus, negatively affect seed resistance. An accomplishment of germination (Tylkowska et al. 2003, new varieties of these crops exhibiting Bart & Thomma 2003). Moreover, resistance against the disease is re- A. brassicae- and A. brassicicola- garded as potentially most economi- produced cause green dis- cally feasible solution to limiting the colorations within the diseased spots yield losses. Such a task remains cru- (Tylkowska et al. 2004). A. brassicae- cial from the standpoint of the vegeta- derived abscisic acid causes prema- ble producers, as well as of the con- ture leaf aging and defoliation, drop- sumers. It allows for a decrease in ping flowers, or premature breaking pesticide use, which is of particular of the siliques (Tewari 1991a). importance in the integrated and eco- logical vegetable production. Unfor- Disease impact tunately, such resistance breeding of The highest toll the Alternarias the crops is currently hindered due to collect is on the seed plantations of bottlenecks experienced in transfer of the oleraceous Brassicas, including resistance from the wild species into the cabbages. Infected silique tissue commercial lines. perishes and withers, as a result of Until now, no high-level re- which the siliques shrink, break open, sistance sources against A. brassicicola and the seeds drop (Maude & or A. brassicae have been identified Humpherson-Jones 1980), which gen- among the cultivated species of the erates significant economic losses. Brassica genus; however, individual Upon strong infections of young si- varieties among the cabbages may liques, the seeds do not develop, or differ in the exhibited levels of sus- remain underdeveloped, and exhibit ceptibility to black spot (Otani et al. decreased vigor and germinability 2001). The highest level of Alternaria (Chirco & Harman 1979). Infected resistance from among the Brassica seeds may display the hyphae present crops is displayed by the Ethiopian on their surface (surface infection), mustard (B. carinata). Among the wild but the hyphae is able to grow through cruciferous plants closely related to the the seed cover (internal infection). Brassica genus, the highest Spores localized both internally and A. brassicae resistance levels were externally may survive several years, confirmed for white mustard (Sinapis although the internal infection seems alba; Kolte 1985, Brun et al. 1987, to be more durable (Maude & Ripley et al. 1992, Sharma & Singh Humpherson-Jones 1980). Seedlings 1992, Hansen & Earle 1995, 1997); developing from infected seeds show however, the highest overall Alternaria typical symptoms of damping off spp. resistance has been identified in (small black spots on the bottom leaf the crucifers species more distant from surface or dark stripes on the hypo- the Brassica, such as camelina (Came- cotyls).

12 VEGETABLE CROPS RESEARCH BULLETIN 76 ______lina sativa; false flax), shepherd's- epicuticular wax layer forming a hy- purse (Capsella bursa-pastoris), rucola drophobic coating to reduce the ad- (Eruca sativa), and ball mustard herence of water-borne inoculum, as (Neslia paniculata) (Conn & Tewari well as limiting spore germination 1986, Conn et al. 1988, Tewari rate (Meena et al. 2010). Presence of 1991b). Resistance against Alternaria intensive leaf wax deposition seems black spot has also been reported correlated with the resistance exhibited among other wild members of the by other Brassicacae plants (Meena et Brassicacae family (Sharma et al. al. 2010). 2002, Tewari & Conn 1993; reviewed Wild crucifers are found to elicit and referenced in Warwick 2011): phytoalexins upon challenge inocula- Alliaria petiolata; Barbarea vulgaris; tion (Conn et al. 1988). Among the Brassica elongate, B. desnottessi, Alternaria-resistant species, camelina B. fruticulosa, B. maurorum, B. nigra, stands out for its immunity against B. souliei, B. spinescens; Camelina A. brassicicola infection, originating sativa; Capsella bursa-pastoris; Coin- in the plant’s ability to synthesize cya spp.; Diplotaxis catholica, camalexin, a compound with antibi- D. berthautii, D. creacea, D. erucoides, otic properties, and thus to hamper D. tenuifolia; Erucastrum gallicum; pathogen’s development. Indeed, it Eruca vesicaria subsp. sativa; Hemi- has been demonstrated that camalexin crambe fruticulosa, H. matronalis; deficient Arabidopsis mutant, pad-3 is Neslia paniculata; Rhaphanus sativus; more susceptible to A. brassicicola S. alba, and S. arvensis. The com- than wild-type plants (Zhou et al. pletely-immune plants remained symp- 1999). Additional evidence that tom-free both, under natural field in- camalexin plays a major role in re- fection, as well as under controlled sistance came from the observation artificial inoculation (Sharma et al. that different Arabidopsis ecotypes 2002). Comparatively, broccoli and with varying levels of camalexin cauliflower varieties exhibited only show correlative differential re- moderate Alternaria resistance, while sistance (Kagan & Hammerschmidt the cabbages turned out susceptible. 2002). Finally, the esa1 mutation af- fects resistance against A. brassicicola Resistance background through a severe reduction in both Depending on the plant material camalexin production, as well studied, A. brassica/A. brassicicola jasmonate-depen-dent gene induction, resistance was said to be controlled by although the Esa1 gene has yet to be one or several nuclear genes of par- cloned (Tierens et al. 2002). tially-dominant interaction (Zhang et al. 1997) or is conditioned by additive Resistance testing inheritance (Krishnia et al. 2000). On A direct method of determination the biochemical level, resistance of Alternaria resistance are the phyto- against Alternaria pathogens seems to pathological tests: Field-, greenhouse- be connected with high activities of , or phytotron-based. Field observa- phenolases (polyphenol oxidase, pe- tions can be carried out upon natural roxidase, catalase), high levels of leaf pathogen infection, or after controlled sugars (Singh et al. 1992), and thicker artificial inoculation with fungal spore

M. NOWICKI et al. – ALTERNARIA BLACK SPOT … 13 ______suspension. Advantages of the green- sides on the adaxial leaf surface, due house or phytotron tests are: Speedi- to which the aqueous spore suspen- ness, reproducibility, and a possibility sion gets uniformly distributed on the of control of the conditions. Phyto- leaf surface, without the need of add- pathological tests require Alternaria ing agar or adjuvants (Sharma et al. spp. conidia, collected directly from 2002). Better adhesion of the water- the infected plant tissue or maintained suspended spores to wax-covered leaf on the artificial media. On the com- surface of cabbages is granted by monly used PDA artificial media, addition of agar (Ho et al. 2007) or fungal growth and effective spontane- Tween (Doullah et al. 2006). Thin ous sporulation take place at 25±2ºC, needle has been employed to make in darkness. A choice of other meth- small surface cuts, onto which a drop- ods exist towards Alternaria spp. let of inoculum (4×103 spores×ml-1) growth and maintenance. A. bras- was placed. sicicola hyphae has been successfully Disease symptoms have been cultured on artificial media V8A (V8 scored in 24 h increments for 3 dpi. juice - agar) at 25ºC, resulting in Resistance scoring of individual plants spontaneous sporulation under 12 h included three parameters: Percentile photoperiod (Otani et al. 1998). of infected leaf surface (0-60 pts), le- Controlled-conditions phyto- sion size (0-30 pts), and incubation tests are carried out on whole plants duration (0-10 pts). Plants exhibiting (in vivo) or on detached leaves (in maximum susceptibility scored vitro; Sharma et al. 2002). Plants are 100 points. Individual plants have been routinely tested at 3-6 weeks seed- grouped into the resistance classes, lings stage, but cotyledon phyto-tests according to their points scoring: 0- have been published as well (Doullah fully resistant; 1-15 pts – moderately et al. 2006). The detached leaf method resistant; 16-25 pts – susceptible; is one of the most often employed above 25 pts – highly susceptible. As ways to assess the Alternaria spp. discussed above, optimal phyto-test resistance levels displayed by the conditions are temperatures of about tested plants under controlled condi- 20○C, relative humidity of at least 90% tions. Differences exist, however, lasting for 6 h and more, and inoculum regarding the inoculation method and load of 6×104 spores×ml-1 (Sharma et assay conditions. As described in al. 2002, Doullah et al. 2006). several studies, inoculum of 5×104 Bottlenecks in resistance breeding spores×ml-1 was placed at the upper Since resistance against Alter- (adaxial) leaf side (Doullah et al. naria black spot is generally governed 2006), while other authors described by polygenes, breeding for resistance spraying the lower (abaxial) leaf side could involve pyramiding of minor with inoculum of 3×105 spores×ml-1 genes to provide additive/polygene (Parada et al. 2008). Yet others inocu- resistance. Rapid advances in tech- lated only the 4th and 5th leaves (seed- niques of tissue culture, protoplast ling 45 days old; Sharma et al. 2002). fusion, embryo rescue, and genetic Wet swabs have been used to remove engineering have made possible the the leaf wax layers on both nerve transfer of disease resistance traits

14 VEGETABLE CROPS RESEARCH BULLETIN 76 ______across the otherwise impassable self- to this of S. alba, B. oleracea var. incompatibility barriers. Transgenic botrytis, or B. carinata (Ryschka et al. plants with disease resistance which 1996). Seeds of developed intertribal over-express different antifungal com- somatic hybrids between B. napus and pounds like pathogenesis-related (PR) C. sativa (by means of protoplast elec- proteins (chitinase, glucanase, osmotin, trofusion) exhibited phenotype inter- etc.) and ribosome inhibiting proteins mediate compared with the parental (RIPs) such as thionins, defensins, and species. They also exhibited higher phytoalexins (Zhou et al. 2002) to level of linolenic and eicosanoic acids, inhibit growth of the pathogen, seem but the hybrid plants await determina- less efficacious. tion of their Alternaria resistance To introduce camelina-derived (Jiang et al. 2009). A. brassicicola resistance into com- In general, it has been postulat- mercial varieties, somatic hybrids ed, that introduction of Alternaria between C. sativa and B. carinata resistance genes into commercial cul- have been procured; however, the tivars of crucifers is dependent on researchers failed to multiply the re- cumulation of horizontal resistance sulting hybrids (Narasimhulu et al. genes (Sharma et al. 2002). Hence, it is 1994). Similar strategy of protoplast imperative to identify various sources fusion between C. sativa and of horizontal resistance among the B. oleracea with subsequent hybrid Brassica plants (see above), and sub- regeneration also proved unsuccessful sequently to combine them towards (Hansen 1998). Several research increase in durable Alternaria protec- groups attempted, but not succeeded, tion. Strong cross-incompatibility, to introduce the E. sativa-deriving polygenic background of the re- black spot resistance into various spe- sistance (additive and dominant gene cies of cultivated crucifers (Fahleson interactions), as well as the differ- et al. 1988, Sikdar et al. 1990, Siga- ences in ploidy (differing number of reva & Earle 1997). The first somatic chromosomes) between respective hybrids to be obtained as a result of Brassicaceae species render the trans- protoplast fusion were those of fer of Alternaria resistance from the B. napus () and S. alba (Pri- wild species into the cultivated forms mard et al. 1988). None of the hybrids difficult. Additionally, it is often con- procured that way showed nected with employment of advanced A. brassicae resistance comparable to in vitro hybridization techniques, in- that exhibited by S. alba. Chevre et al. cluding somatic hybridization, embryo (1991) used these species towards and ovary rescue, or protoplast fusion. interspecies crosses through somatic hybridization and bidirectional cross- Acknowledgements es. Having employed the embryo res- Authors are indebted to Dr. cue technique, the researchers suc- Wanessa Wight, PhD (Michigan State ceeded in regeneration of B. napus University, U.S.) for critical reading of plants carrying 38 chromosomes typi- the manuscript. Alternaria resistance studies in the E.U. Kozik’s lab were sup- cal for that species, and displaying ported by the Polish Ministry of Agricul- A. brassicae resistance at levels close

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ALTERNARIOZA ROŚLIN KAPUSTOWATYCH: OBJAWY, SZKODLIWOŚĆ I PERSPEKTYWY HODOWLI ODMIAN ODPORNYCH

Streszczenie Alternarioza kapustowatych (syn. czerń krzyżowych, czarna plamistość roślin krzyżowych) powoduje duże straty gospodarcze w wielu krajach, w tym również w Polsce. Sprawcami choroby są różne gatunki grzybów z rodzaju Alternaria, najczę- ściej: A. brassicae (Berk.) Sacc., A. brassicicola (Schw.) Wiltsh., A. raphani Groves i Skolko oraz A. alternata (Fr.) Kreissler. Roślinami żywicielskim są kapusty i inne rośliny uprawne oraz dziko rosnące z rodziny krzyżowych. Szkodliwość czerni krzyżo- wych w uprawie kapusty głowiastej i pekińskiej polega na obniżeniu wysokości i jako- ści plonu. Grzyby z rodzaju Alternaria wywołują również zgorzel siewek, zgorzel pod- stawy główek kapusty, brązowienie róż kalafiora oraz brokułu. Najczęściej porażane organy wegetatywne roślin kapustowatych są infekowane przez A. brassicicola i A. brassicae, natomiast w uprawie nasiennej roślin oleistych w obrębie rodzaju Bras- sica dominującym sprawcą alternariozy jest A. brassicae. Pierwotnym źródłem choroby są głównie zakażone nasiona, ale także zimotrwałe rośliny z rodziny krzyżowych, reszt- ki porażonych roślin, a na plantacjach nasiennych również materiał wysadkowy.

M. NOWICKI et al. – ALTERNARIA BLACK SPOT OF CRUCIFERS … 19 ______Metody zapobiegania i zwalczania alternariozy na plantacjach polegają na łącze- niu zabiegów agrotechnicznych z ochroną chemiczną. Podstawową metodą zapobiega- nia chorobie jest produkcja zdrowych nasion, które otrzymuje się, stosując systema- tyczne opryskiwanie plantacji nasiennych fungicydami. W pierwszym roku uprawy zwalczanie ogranicza się do przedsiewnego zaprawiania nasion, a w czasie wegetacji, w okresach wzmożonego zagrożenia chorobą, znaczne obniżenie porażenia uzyskuje się dzięki regularnym opryskom środkami grzybobójczymi. Według wielu autorów, naj- bardziej ekonomicznym rozwiązaniem byłoby uzyskanie odpornych odmian warzyw kapustowatych. Pozwoliłoby to na zmniejszenie zużycia pestycydów, co ma szczególne znaczenie w uprawach integrowanych i ekologicznych. Pomimo identyfikacji źródeł odporności wśród roślin z rodziny krzyżowych, przeniesienie tej cechy do uprawnych gatunków kapustowatych jak dotąd nie powiodło się. Silne bariery niezgodności krzyżowej, poligeniczne uwarunkowanie odporności (addytywne i dominujące współdziałanie genów) oraz różnice w ploidalności pomiędzy poszczególnymi gatunkami rodziny krzyżowych sprawiają, że przeniesienie genów odporności z dzikich gatunków do form uprawnych jest bardzo trudne i wymaga wyko- rzystania technik hybrydyzacji in vitro (w tym: somatyczna hybrydyzacja, „embryo/ ovary rescue”, fuzja protoplastów). Jednak dzięki coraz lepszemu poznaniu wzajem- nych relacji patogen-roślina żywicielska, identyfikacji nowych źródeł odporności oraz określeniu mechanizmu dziedziczenia tej cechy, możliwy będzie postęp w hodowli roślin kapustowatych odpornych na alternariozę.