Studia Universitatis 3.Indd

Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii Vol. 21, issue 3, Jul.-Sep. 2011, pp. 593-598 ©2011 Vasile Goldis University Press (www.studiauniversitatis.ro) INDUCED IN VITRO CO-CULTURE BETWEEN ASPARAGUS OFFICINALIS L. PHYTOINOCULA AND A SAPROPHYTIC FUNGUS

Adriana Petruş-Vancea*1, Dorina Cachiţă-Cosma2, Violeta Turcuş2, Iudith Ipate3, Cornelia Purcărea1, 1 University of Oradea and postdoctoral researcher at ”Vasile Goldis” West University of Arad, Romania 2”Vasile Goldis” West University of Arad, România 3 Romanian Academy, INCE, Center for Studies and Researchers Agroforestry Biodiversity, Bucharest, România

ABSTRACT. The aim of present research is to investigate the nature of the relationship established among partners, forced associates, depending on ecophysiological conditions, namely induced in vitro co-culture between Cladosporium sp. fungus and Asparagus officinalis L. phytoinocula. At 30 days from co-culture initiation, on Murashige-Skoog mineral medium, without growth regulators, lightened with white fluorescent light, 1700 lx intensity, with the photoperiod of 16 h day length/24h, and a temperature of 20 ± 2ºC, the two partners no interacted, and both had normally developed. Asparagus explants provided from a co-culture with Cladosporium sp. which was individual cultivated on fresh medium, ulterior showed no infection. Keywors: fungus, Asparagus, co-culture, micropropagation

INTRODUCTION in this area. This method have more advantages: using Plant biotechnology will be the domain that, in the of sterile host in aseptically conditions; small tissue future, will provide much needed human nutrition, in used to reduce the space needed; the size of inocula, the addition to traditional methods (Antofie et al., 2010 a pathogen penetration mode and rapid assessment can be and b). In vitro cultures are used for many plant species, done, deliberate manipulation of physical and chemical which enables us to obtain healthy plants in a short time. environment and no unnecessary risk of introduction the In practice there are, however many difficulties connected pathogen to the field (Nik and Rahman, 1993). with the obtaining of regenerative active and viruses - In natural conditions, root rot in Asparagus caused free explants (Cassells, 1991; Cooper et al., 2006). by two species of Fusarium (Fusarium oxysporum f.sp. One of the problems is the infection of phytoinocula asparagi and Fusarium proliferatum) is present in many caused by fungi, drawing into the elimination of cultures (Lewis and Shoemaker, 1964; Dan and Stephens, explants from further micropropagation techniques. The 1995), being the major cause of asparagus disease with results of works done so far Leifert and collaborators great loses in commercial planting of asparagus and other (1994), Debergh (1996), Leifert and Woodward (1997), varieties of Fusarium oxysporum affect cabbage, radish, Williamson and collaborators (1997) present a wide carnation, cotton, melon, potato and tomato (Davis, 1969), variety of fungi species, appearing in the in vitro cultures, Pinus resinosa (Farquar and Peterson, 1989) or banana on the subsequent stages of micropropagation. (Matsumoto et al, 2010). This fungus produced toxins About thirty-one microorganisms from ten different like fusaric acid (FA), lycomarasmin, moniliformin, plant cultivars growing in micropropagation have been naphthazarin, sambucin and beauvericin (Švábová and isolated, identified and characterized, with Yeasts, Lebeda, 2005) and the chemical treatment is no effective. Corynebacterium spp. and Pseudomonas spp. being Cassini et al. (1985) employed an antagonist microbe to predominant (Leggatt et al., 1994). control Fusarium infections of field-grow asparagus. On the other hand, in the present, in modern plant There are antifungal antibiotics from actinomycete, biotechnology, there is a new scientific branch, a “hybrid” like faerie fungi which were used in asparagus culture, between biotechnology focused on phytoinocula and that without adversely affect (Smith et al., 1990). This focusing on fungi, namely the co-culture (associated antibiotic at 25 ppm decreased the severity of disease in culture) (Cachiţă et al., 2008; Turcuş and Cachiţă, 2009), sterile asparagus plant grown on water agar and increased purpose of establishing symbiosis, raising productivity fibrous root weight of plants grown in the absence of F. of plants, especially fruit and forest species or increase oxysporum. The producer organism, grown on several the synthesis of secondary metabolites, or the selection media, found to be antagonistic to F. oxysporum. In the of plants to certain pathotoxin resisted etc. (Cachiţă and other hand, many papers and reviews have discussed the Ardelean, 2009). advantages of using chitinases for plant protection because The tissue cultures can be used for studying host- these enzymes are fungicidal, part of the plant defense pathogen interaction (Ingram and Helgeson, 1980; Devi system, and nontoxic to plants, but Cladosporium fulvum, and Srinivasan, 2006), supplementing the screening Cercospora nicotiana, C. beticola, Fusarium solani, and field, helping to accelerate the assessments and studies several other pathogenic fungi were not inhibited in the

*Correspondence: Adriana Petrus-Vancea, University of Oradea, Faculty of Science, Biology Department, no 1, Universitatii St., Oradea, Bihor, Romania, 410082, E-mail: [email protected] Petruş-Vancea A., Cachiţă-Cosma D., Turcuş V., Ipate I., Purcărea C. growth by treatment with a enzyme namely chitinases solidified Murashige-Skoog (MS) (1962) medium, obtained from plant (Joosten et al., 1995). without growth regulators or aminoacids, with 20 g/l Also, genus of Asparagus can be infected by sucrose. The culture was incubated at 22 - 23ºC, at 1700 Stemphylium botriosum a fungus which affect cladodes, lux light intensity, generated by white fluorescent lamps, branches and the main stem, with defoliation (Bansal with 16/24 light photoperiod, during 8 weeks. After that, et al, 1986) or by Puccinia asparagi that causes rust on the 3 cm long plantlets were transferred to fresh same MS Asparagus, or Phytophtora ssp., Pleospora herbarum media, kept in vitro for other 12 weeks. (Koike et al., 2007). The number of culture recipients was 400. Just in one Rahman and collaborators (1987) used the in vitro recipient, at 6 week after inoculations, the spontaneous method on conifer adventition shoots inoculated with infection appeared. The infection increased, the fungus Gremmeniella abietina to elucidate host-pathogen developed a like-velvet mycelium, with gray colour, interaction. whose hyphae no invading culture medium deep, and in These studies come to indicate a possible positive 12 weeks had a fructification body (Fig. 1), looking like effect could be having the contamination of in vitro plant a crater, about 1.5 cm height, with a wall about 3 mm, cultures with a saprophytic fungus. with dense structure of hyphae and numerous spores. In other instances, a fungal infection of such scale could be MATERIAL AND METHODS smothered phytoinocula. Paradoxically, not revealed any Asparagus minicuttings from the greenhouse, with incompatibility between fungus and phytoinocula, both 3 cm length was surface sterilized with 4 % sodium have a long extending, without interacting, so that at 6 hypochlorite (v/v). Then the fragments were rinsed 5 weeks after initial spontaneous co-culture (12 week after x with sterile water for 5 minutes each. After necrosis inoculation) we proceed to subculture after protocols removal 1 cm fragments, with nodes, was placed on from Table 1:

Table 1. The protocol of second induced co-culture. Type of phytoinocula Culture media Culture period CC-Asparagus shoots (1 cm length) and fungus MS solidified, mycelium (0.5/0.5 cm) without growth 30 days AC-Asparagus shoots (1 cm length) regulators FC-Fungus mycelium (0.5/0.5 cm) Note: CC – co-culture; AC – Asparagus culture; FC – fungus culture.

This subculture was made in culture vessel with 2 covered the culture vessel. This hypothesis was rejected by cm diameter and 7 cm height. a test which used a lot of vitroculture which was covered Assessment was carried out as 30 days after second with this type of film, and another identical blocked co-culture initiation. by aluminium foil (which were initially autoclaved the From fructification body was prelevated fragment vessel, so it was sterilized). At 6 weeks after inoculation which was observed at optical microscope Leitz brand, in these conditions of covering, infection occurred in both Webster M and the most representative images were groups. Other possible causes of infection occurrence taken with an adapted digital camera. may be: improper sterilization of culture medium, culture vessel, instrumentation necessary for inoculation or a RESULTS AND DISCUSSIONS error in technique, but in all these cases, the occurrence of Fungus identified by us is identical to that described infection should be in the first 5-7 days after inoculation. by Cachiţă and collaborators (2008 a and b), respectively Another hypothesis would be the existence of an ill plant one species of the genus Cladosporium. If at that time, material in earlier vitroculture, but it is not supported the above mentioned authors stated that at Drosera by the fact that, from a bush located in vitroculture, the rotundifolia L. was highlighted this allelopathy lack propagules was multiplicated in 10 new vessels, and our of response, explained that this is a carnivorous plant, new infection was singular. Probably, the fungus spores in these studies can prove that the species of the genus are in the growth room and penetrate the polyethylene Asparagus (monocotyledonous plants) have the same co- film in its sensitive areas. Such a situation has worried us culture tolerance with a fungus. very much, because there is a risk of a pandemic. Since, the occurrence of the infection (Fig. 1 A and B) was after the discharge a considerable time after in vitro culture initiation, we considered that the cause of this phenomenon was polyethylene foil with which we

594 Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii Vol. 21, issue 3, Jul.-Sep. 2011, pp. 593-598 ©2011 Vasile Goldis University Press (www.studiauniversitatis.ro) Induced in vitro co-culture between Asparagus officinalis L. phytoinocula and a saprophytic fungus

Fig. 1. Spontaneous appearance of Cladosporium sp. fungus in Asparagus officinalis L. vitroculture, after 12 week of inoculation: A, B – in vitro co-culture between phytoinocula and fungus, C- wall of fructification body detail, D – Asparagus vitroplantlets provided from spontaneous co-culture (arrow means the fungus fructification body; bars means 1 cm) (original).

OdutayoOdutayo andand collaboratorscollaborators (2007)(2007) investigatedinvestigated the sourceAt of Asparagus microbial, co-culturecontamination phenomenon in tissue has culture been laboratories, in south-western Nigeria. They observed nineteen microbial contaminants (consisting of eleven the source of microbial contamination in tissue culture reported by the Cachiţă and collaborators (2008), but the bacteria and eight fungi) were found associated with the tissue culture plants and the laboratory laboratories, in south-western Nigeria. They observed authors mentioned studied just only co-culture occurred environments. The rate of occurrence of Streptococcus aureus, Bacillus cereus, B. subtilis and Escherichia nineteen microbial contaminants (consisting of eleven in vitro spontaneously, not made subcultures of plant and coli were found to be higher (ranging from 36-46%) in human skin than in all other sampled materials. The laboratorybacteria and walls eight fungi)and tables were foundalso haassociatedrboured with most the of the contaminatingmicrobial material, microbes. from a alsoThe co-culture.laboratory We indoor realized air wastissue found culture associated plants and with the all laboratory the contaminating environments. microbes. this co-culture in order to demonstrate that, after 6 weeks, TheOmamor rate of occurrence and co-workers of Streptococcus (2007) identifiedaureus, Bacillus twenty-five thespecies phytoinocula of fungi does belonging not get infectedto 14 genera with fungus, as fungal even contaminantscereus, B. subtilis of theand oilEscherichia palm tissue coli culturewere found materials to be (explant,if it formed callus/embroid a fructification and plantlets).body, and has Of sporesthese (Fig.13.4 2).% washigher Cladosporium (ranging from sp.36-46%) But, thesein human fungal skin species than in allwere foundWe assumeto cause that death infection of the is cultureinevitable, material. however, Authors but in mentionedother sampled that materials. some Theprobable laboratory source wallss andof contaminationstables the individual such as subculturehandling doesof plantnot occur. materials, culture vesselsalso harboured and the most laboratory of the contaminating were discussed. microbes. The Litz and Conover (1981) mentioned that the laboratoryAt Asparagus indoor air, co-culture was found phenomenon associated with has all beenthe reportedinfection by thewould Cachi developţă and on collaboratorsthe medium later. (2008), Only butthe thecontaminating authors mentioned microbes. studied just only co-culture occurredlatent in vitroinfections spontaneously, may occur notin the made establishment subcultures stage of plantOmamor and microbial and co-workers material, (2007) from aidentified also co-culture. twenty- We realized(Reed et this al., 1995).co-culture in order to demonstrate that, afterfive species 6 weeks, of fungithe phytoinocula belonging to does14 genera not get as fungalinfected with fungus,Analyzing even if the it formed fructification a fructification cone fragments body, and of hascontaminants spores (Fig. of the2). Weoil palmassume tissue that culture infection materials is inevit able,optical however, microscope but in the we individual could highlight subculture the mycelium does not occur.(explant, callus/embroid and plantlets). Of these 13.4 of fungus and sporulated forms (Fig. 2). % wasLitz Cladosporiumand Conover sp. (1981) But, thesementioned fungal thatspecies the were infection wouldA B C develop on the medium later. Only the latent infectionsfound to cause may occurdeath ofin thethe establishmentculture material. stage Authors (Reed et al., 1995). mentionedAnalyzing that somethe fructification probable sources cone of contaminationsfragments of optical microscope we could highlight the mycelium of fungussuch as andhandling sporulated of plant forms materials, (Fig. culture2). vessels and the laboratory were discussed.

Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii 595 Vol. 21, issue 3, Jul.-Sep. 2011, pp. 593-598 ©2011 Vasile Goldis University Press (www.studiauniversitatis.ro) A B C Fig. 2. Cladosporium sp. mycelium and spores, from fructiferous body, at 6 weeks from it spontaneous appearances on first cultures medium (A-C- 200X) (original).

From this spontaneous co-culture, we made an induced co-culture (Fig 3).

Odutayo and collaborators (2007) investigated the source of microbial contamination in tissue culture laboratories, in south-western Nigeria. They observed nineteen microbial contaminants (consisting of eleven bacteria and eight fungi) were found associated with the tissue culture plants and the laboratory environments. The rate of occurrence of Streptococcus aureus, Bacillus cereus, B. subtilis and Escherichia coli were found to be higher (ranging from 36-46%) in human skin than in all other sampled materials. The laboratory walls and tables also harboured most of the contaminating microbes. The laboratory indoor air was found associated with all the contaminating microbes. Omamor and co-workers (2007) identified twenty-five species of fungi belonging to 14 genera as fungal contaminants of the oil palm tissue culture materials (explant, callus/embroid and plantlets). Of these 13.4 % was Cladosporium sp. But, these fungal species were found to cause death of the culture material. Authors mentioned that some probable sources of contaminations such as handling of plant materials, culture vessels and the laboratory were discussed. At Asparagus, co-culture phenomenon has been reported by the Cachiţă and collaborators (2008), but the authors mentioned studied just only co-culture occurred in vitro spontaneously, not made subcultures of plant and microbial material, from a also co-culture. We realized this co-culture in order to demonstrate that, after 6 weeks, the phytoinocula does not get infected with fungus, even if it formed a fructification body, and has spores (Fig. 2). We assume that infection is inevitable, however, but in the individual subculture does not occur. Litz and Conover (1981) mentioned that the infection would develop on the medium later. Only the latent Petruş-Vanceainfections may A., occurCachiţă-Cosma in the establishment D., Turcuş V., Ipate stage I., Purcărea (Reed etC. al., 1995). Analyzing the fructification cone fragments of optical microscope we could highlight the mycelium of fungus and sporulated forms (Fig. 2).

A B C Fig. 2. Cladosporium sp. mycelium and spores, from fructiferous body, at 6 weeks from it spontaneous appearances on first cultures medium (A-C- 200X) (original).

From this spontaneous co-culture, we made an induced co-culture (Fig 3).

Fig. 3. Aspects of induced co-culture (A), of individual Asparagus phytoinocula subculture provided from spontaneous Fig.co-culture 3. Aspects (B) and of induced individual co-culture fungus subculture(A), of individual (C), in Asparagus the first day phytoinocula of second induced subculture co-culture provided (bars from means spontaneous 1 cm) co-culture(original). (B) and individual fungus subculture (C), in the first day of second induced co-culture (bars means 1 cm) (original). AtAt 30 days after initiation of induced induced co-culture, , each havingof the threean average types 2.5of culture cm in hassize, developed which shows very that well each(Fig.At of 304).the daysThus,three typesafter induced ofinitiation culture co-culture ofhas induced developed between co-culture very Asparagus well, each phytoinocula ofalthough the three it originate andtypes fungal of fromculture mycelium, a co-culturehas developed both with grew a very normallyfungus, well (Fig.(Fig.without 4).4). Thus, Thus,interaction, induced induced without co-culture co-culture affecting between between each Asparagus Asparagusother (Fig. phytoinocula4 phytoinoculaA). Culture and ofnot individual fungal disease mycelium, Asparagusmanifests, both ,being provided grew grown normally from as a phytoinoculawithoutspontaneous interaction, and co-culturefungal without mycelium, not affecting has both anyeachgrew form normallyother of (Fig. infe 4ction, A).individuals Culture the regeneration of(Fig. individual 4 B). InprocessAsparagus turn, cultivatedwas, provided complete, individual from the a withoutspontaneousvitroplantlets interaction, havingco-culture without an averageaffectingnot has 2.5each any cm other formin size,(Fig. of which4infe ction, shfungusows the that showed regeneration although a normal it originat growth,processe reaching fromwas acomplete, co-culture1 cm diameter thewith A).vitroplantletsa Culturefungus, ofphytoinocula havingindividual an Asparagusaverage not disease 2.5, provided cm manifests, in size, from which abeing sh ofgrownows about that as a although month,individuals as it well originat (Fig. as ite4 from fromB). co-culture,In a co-cultureturn, cultivated both with in a fungus, phytoinocula not disease manifests, being grown as individuals (Fig. 4 B). In turn, cultivated spontaneousindividual fungusco-culture showed not has a normalany form growth, of infection, reaching 1 cmterms diameter of size andof about shape aand month, macroscopic as well aspects as it from (Fig. co-4 individual fungus showed a normal growth, reaching 1 cm diameter of about a month, as well as it from co- theculture, regeneration both inprocess terms was of size complete, and s thehape vitroplantlets and macroscopic C). aspects (Fig. 4 C). culture, both in terms of size and shape and macroscopic aspects (Fig. 4 C).

Fig. 4. Aspects of induced co-culture (A), of individual Asparagus phytoinocula subculture provided from spontaneous Fig.co-culture 4. Aspects (B) and of induced individual co-culture fungus subculture(A), of individual (C), at Asparagus 30 day of second phytoinocula induced subculture co-culture provided (bars means from spontaneous1 cm) (original). co-culture (B) and individual fungus subculture (C), at 30 day of second induced co-culture (bars means 1 cm) (original). In 1990, Nik and Salleh, on Asparagus tissue culture inoculated with Fusarium proliferatum, studied the surfaceIn 1990, activity Nik andto the Salleh, fungus on on Asparagus the host and tissue its modeculture of inoculated penetration. with The Fusarium authors usedproliferatum filtered, suspensionsstudied the surfaceand cultures activity of to Fusarium the fungus oxysporum on the host. Inoculation and its mode with of conidia penetration. led to The the authorshyphae usedformation filtered in suspensionsless than 24 andhours, cultures in two of days Fusarium there wasoxysporum a distinct. Inoculation network of with hyphae conidia on tledhe surfaceto the hyphae tissue. Atformation 6 days Asparagusin less than stem 24 hours,necrosis in twooccurred, days there and atwas 12 a days distinct it was network spread of hyphaethroughout on thethe surface stem. In tissue. some At cases, 6 days at Asparagusthe 8th day stem was 596necrosisdeveloped occurred, a large and mycelium at 12 days on the it was stem spread surface, throughout but without theStudia stem.the Universitatis appearance In some “Vasile cases, of Goldiş”, necrosis. at theSeria 8th ŞtiinţeleHowever, day Vieţiiwas in developed a large mycelium on the stem surface, but without the appearanceVol. 21, issue of 3, Jul.-Sep.necrosis. 2011, However, pp. 593-598 in transversal section trough stem were observed red formations©2011 Vasile inGoldis subepidermal University Press layers. (www.studiauniversitatis.ro) Fusarium hyphae transversalpenetration section occurred trough directly stem through were theobserv layersed redof epidermi formationss rarely, in subepidermal because there layers. is the Fusarium cuticle layer hyphae (Nik penetrationand Salleh, occurred 1990). In directly fact, cracks, through stomata the layers openings of epidermi provideds rarely, a quick because access tothere underlying is the cuticle tissues. layer Infection (Nik andalso Salleh, caused 1990). dark Indeposits fact, cracks, in the stomata vascular openings system toprov form,ided thusa quick leads access to wilting. to underlying Inoculation tissues. of Asparagus Infection alsoshoots caused with maturedark deposits filtered tissuein the culturevascular led system to chlo rosesto form, and thus death leads of infected to wilting. plant Inoculation in less than of fourAsparagus days. shoots with mature filtered tissue culture led to chloroses and death of infected plant in less than four days.

CONCLUSIONS CONCLUSIONSGrowing Asparagus officinalis L. phytoinocula and a fungus in in vitro co-culture conditions, did not affect theGrowing two species, Asparagus neither officinalis positive norL. phytoinocula negative. and a fungus in in vitro co-culture conditions, did not affect the twoThe species, Asparagus neither officinalis positive L. phytoinoculanor negative. was not infected by fungus, in co-culture conditions. TheFor Asparagusin vitro micropopagation officinalis L. phytoinocula the occurrence was of not such infected infections by fungus, can lead in co-culture to large losses conditions. for producers, but in Forthis in case, vitro micropopagationwe could prove thethat occurrence the fungus of doessuch infectionsnot affect canthe leadproduction to large of losses biomass, for producers, as described but inexperiment, this case, itwe can could be studied prove in that future the in fungusorder to does achieve not symbiosis.affect the production of biomass, as described experiment, it can be studied in future in order to achieve symbiosis.

ACKNOWLEDGEMENTS ACKNOWLEDGEMENTSThis work was cofinanced from the European Social Fund through Sectoral Operational Programme HumanThis workResources was cofinanced Development from 2007-2013, the European project Social number Fund POSDRU/89/1.5/S/63258 through Sectoral Operational ”Postdoctoral Programme school Humanfor zootechnical Resources biodiversity Development and 2007-2013, food biotechnology project number based POSDRU/89/1.5/S/63258 on the eco-economy and ”Postdoctoral the bio-economy school forrequired zootechnical by eco-sanogenesys”, biodiversity and coordinatedfood biotechnology by the Natibasedonal on Institute the eco-economy of Economic and Research the bio-economy ”Costin C. requiredKiriţescu”, by Romania, eco-sanogenesys”, in collaboration coordinated with ”Lucian by the Blaga” Nati onalUniversity Institute of Sibiu,of Economic ”Vasile Goldi Researchş” West ”Costin University C. KiriArad,ţescu”, S.C. Romania, GNIR Holding in collaboration S.A. and S.C. with SIAT ”Lucian S.A. Blaga” University of Sibiu, ”Vasile Goldiş” West University Arad, S.C. GNIR Holding S.A. and S.C. SIAT S.A.

Induced in vitro co-culture between Asparagus officinalis L. phytoinocula and a saprophytic fungus

In 1990, Nik and Salleh, on Asparagus tissue of crop landraces in Romania. Analele Universităţii culture inoculated with Fusarium proliferatum, studied din Oradea – Fascicula Biologie, XVII (2), pp 313- the surface activity to the fungus on the host and its mode 317. of penetration. The authors used filtered suspensions Bansal R.K, Menzies S.A, Broadhurst P.G. (1986). and cultures of Fusarium oxysporum. Inoculation with Screening of Asparagus species for resistance to conidia led to the hyphae formation in less than 24 hours, Stemphylium leaf spot. New Zealand Journal of in two days there was a distinct network of hyphae on Agricultural Research, 29, pp. 539-545. the surface tissue. At 6 days Asparagus stem necrosis Cachiţă C.D, Ardelean A. (2009). Cocultura sau culturile occurred, and at 12 days it was spread throughout the asociate dintre fitoinoculi şi microorganisme. stem. In some cases, at the 8th day was developed a large In: Cachiţă CD, Ardelean A (eds),Tratat de mycelium on the stem surface, but without the appearance biotehnologie vegetală, Vol. II, Edit. Dacia, pp. 87- of necrosis. However, in transversal section trough stem 170. were observed red formations in subepidermal layers. Fusarium hyphae penetration occurred directly through Cachiţă C.D, Turcuş V, Hurgoiu F, Petruş-Vancea A. the layers of epidermis rarely, because there is the cuticle (2008 a). Coculturi de durată produse spontan, layer (Nik and Salleh, 1990). In fact, cracks, stomata între fitoinoculi şi un fung saprofit. In: Cachiţă openings provided a quick access to underlying tissues. CD, Brezeanu A, Ardelean A (eds.), Lucrările celui Infection also caused dark deposits in the vascular system de al XVI – lea Simpozion Naţional de Culturi de to form, thus leads to wilting. Inoculation of Asparagus Ţesuturi şi Celule Vegetale, Bucureşti, intitulat shoots with mature filtered tissue culture led to chloroses ”Biotehnologii vegetale pentru sec. XXI”, Edit. and death of infected plant in less than four days. Risoprint Cluj – Napoca, pp. 185-193. Cachiţă C.D, Turcuş V, Petruş-Vancea A, Barbu-Tudoran CONCLUSIONS L, Crăciun C. (2008 b). Drosera rotundifolia L. Growing Asparagus officinalis L. phytoinocula and vitrocultures associated with a saprophyte fungus. a fungus in in vitro co-culture conditions, did not affect Studia Univ. “Vasile Goldiş”, Seria Şt. Vieţii, 18, pp. the two species, neither positive nor negative. 103 – 106. The Asparagus officinalis L. phytoinocula was not Cassells A.C. (1991) Problems in tissue culture: Culture infected by fungus, in co-culture conditions. contamination. In: Debergh PC, Zinmerman For in vitro micropopagation the occurrence of such RH (eds.), Micropropagation: Technology infections can lead to large losses for producers, but in and Application, Kluwer Academic Publisher, this case, we could prove that the fungus does not affect Dordurecht, Netherlands. Chrissnewell, pp. 31-44. the production of biomass, as described experiment, it Cassini R.C, El Medawar S, Cassini R.P. (1985). A can be studied in future in order to achieve symbiosis. biological control technique to prevent Fusarium decline in the fields. Proceedings of the 6th ACKNOWLEDGEMENTS International Asparagus-Symposium, pp. 228-237. This work was cofinanced from the European Social Cooper C, Crowther T, Smith B.M, Isaac S, Collin Fund through Sectoral Operational Programme Human H.A. (2006). Assesment of the response of carrot Resources Development 2007-2013, project number somaclones to Pythium violae, causal agent of POSDRU/89/1.5/S/63258 ”Postdoctoral school for cavity spot. Plant Pathol., 55, pp. 427-432 zootechnical biodiversity and food biotechnology based on the eco-economy and the bio-economy required by Dan Y, Stephens C.T (1995). The development of eco-sanogenesys”, coordinated by the National Institute asparagus somaclones with high levels of resistance of Economic Research ”Costin C. Kiriţescu”, Romania, to Fusarium oxysporum f. sp. asparagi and F. in collaboration with ”Lucian Blaga” University of proliferatum. Plant Dis. 79, pp. 923-927. Sibiu, ”Vasile Goldiş” West University Arad, S.C. GNIR Davis D. (1969). Fusaric acid in selective pathogenicity Holding S.A. and S.C. SIAT S.A. of Fusarium oxysporum. Phytopathology 59, pp. 1391-1395. REFERENCES Debergh P.C. (1996). Microbial contaminants of plant Antofie MM, Pop, M.R, Sand C, Ciotea G, Iagrăru P. tissue cultures. Agritech Consultans Inc. Skrub Oak (2010 a). Data sheet model for developing a red list USA, pp. 1-75. regarding crop landraces in Romania. Annals. Food Devi SC, Srinivasan M.V. (2006). Studies on various Science and Technology, 11(1), pp. 45-49. atmospheric microorganisms effecting the plant Antofie M.M, Constantinovici D, Pop M.R, Iagrăru tissue culture explants. American Journal of Plant P, Sand C, Cirotea G. (2010ba). Theoretical Physiology 1(2), pp. 205-209. methodology for assessing the status of conservation

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Farquhar M.L, Peterson R.L. (1989). Pathogenesis in Nik N.A, Rahman A. (1993). In vitro inoculation of Fusarium root rot of primary roots of Pinus resinosa asparagus with conidial suspension and culture grown in test tubes. Canadian Journal of Plant filtrate of Fusarium oxysporum schlect f. asparagi. Pathology, 11(3), pp. 221 – 228. In: You CB, Chen Z, Ding Y (eds)., Biotechology in Ingram D.S., Helgeson J.P. (1980). Tissue culture Agriculture, Kluwer Academic Publishers, pp. 336- methods for plant pathologists. Blackwell Scientific 343. Publications, Oxford, UK. Nik N.A, Salleh B. (1990). In vitro inoculation of Joosten MHAJ, Verbakel H.M. (1995). Nettekoven ME, asparagus tissue culture plantlets with vegetative Van Leeuwen J, Van der Vossen TM, De Wit PJGM, hyphae of Fusarium proliferatum. In: Proceeding of The phytopathogenic fungus Cladosporium fulvum 3th International Conference on Plant Protection in is not sensitive to the chitinase and β-1,3-glucanase the Topics, Genting Highlands, Malaysia, pp. 127- defence proteins of its host, tomato. Physiological 129. and molecular plant pathology, 46 (1), pp. 45-59. Odutayo O.I, Amusa N.A, Okutade O.O. (2007). Koike S.T, Gladders P, Paulus A.O. (2007). Vegetable Ogunsanwo YR, Sources of microbial contamination diseases. Elsevier Academic Press, pp. 332-336. in tissue culture laboratories in south-western Nigeria. African Journal of Agricultural Research, Leggatt I.V, Waites W.M, Leifert C, Nicholas J. (1994). 2(3), pp. 67-72. Characterisation Of micro-organisms isolated from plants during micropropagation. In: Bacterial and Omamor B, Asemota A.O, Eke C.R, Eziashi E.I. (2007). Bacteria-like Contaminants of Plant Tissue Cultures Fungal contaminants of the oil palm tissue culture Ishs Acta Horticulturae 225: http://www.actahort. in Nigerian institute for oil palm research (NIFOR). org/books/ 225/index.htm. African Journal of Agricultural Research, 2 (10), pp. 534-537. Leifert C, Woodward S. (1997). Laboratory contamination management: the requirement for microbiological Rahman A, Nik N.A, Diner A.M, Skilling D.D, Karnosky quality assurance. In: Cassells AC (ed.), Pathogen D.F. (1987). In vitro responses of conifer adventitious and Microbial Contamination Management in shoots and calli inoculated with Gremmeniella Micropropagation. Kluwer Acad. Publ., The abietina. Forest Science, 33 (4), pp. 1047-1053(7). Netherlands, pp. 237-244. Reed B.M, Buckely P.M, Dewilde T.N., (1995). Leifert C, Morris C.E, Waites W.M. (1994). Ecology Detection and eradication of entophytic bacteria of microbiological saprophytes and pathogens in from micropropagated mint plants. In vitro cell. tissue culture and fieldgrown plants: Reasons for Dev.Biol., 31, pp. 53-57. Contamination Problems. Critical Rev. Plants Sci. Smith J, Putman A, Nair M. (1990). In vitro control of 13(2), pp. 139-189. Fusarium diseases of Asparagus officinalis L. With a Lewis G.D, Shoemaker P.B., (1964). Resistance of Streptomyces or its polyene antibiotic, faeriefungin. asparagus species to Fusarium oxysporum f. sp. J. Agric. Chem., 38, pp. 1729-1733. asparagi. Plant disease reporter 48,pp. 364-365. Švábová L, Lebeda A. (2005). In vitro selection for Litz R.E, Conover R.A., (1981). Effect of sex type, improved plant resistance to toxin-producing season and other factors on in vitro establishment pathogens. J Phytopathol 153, pp. 52-64. culture of Carica papaya L. explants. J.Am.Soc. Turcuş V, Cachiţă C.D. (2009). Drosera rotundifolia L. Hortic.Sci., pp. 106, 792-794. – particularităţi morfostructurale.”Vasile Goldiş” Matsumoto K, Barbosa M.L, Souza LAC, Teixeira J.B., University Press, Arad, pp. 171-181. (2010). In vitro selection for resistance to Fusarium Williamson B, Cooke DEL, Duncan J.M, Leifert C, wilt in Banana. In: Mass Screening Techniques Breese W.A, Shattock R.C. (1997). Fungal infections for Selecting Crops Resistant to Disease, IAEA of micropropagated plants at weaning: A problem Publishing,Vienna, Austria, pp. 101-109. exemplified by downy mildews in Rubus and Rosa. Murashige T, Skoog F. (1962). A revised medium for In: Cassells AC (ed.), Pathogen and Microbial rapid growth bioassays with tobacco tissue cultures. Contamination Management in Micropropagation. Physiologia Plantarum, 15, pp. 473 – 497. Kluver Acad. Publ., The Netherlands, pp. 309-320.