Current state of genetically modified plant impact on target and non-target fungi
Current state of genetically modified plant impact on target and non-target fungi
F.O.P.Stefani and R.C.Hamelin
Abstract:For two decades,genetic engineering has made it possible to develop crops and trees designed for yield im-provement and simplified culture management.This,combined with field deployment of monocultures over large areas,can result in environmental stress and unwanted potential side effects.The commercial production of genetically modified (GM)crops and the recent development of GM trees raise concerns about their potential impact on the environment,in general,and on the biodiversity of non-target organisms,in particular.Fungi are spread worldwide and play key roles in ecosystems.They have been closely associated with plants since they emerged from the oceans.This review critically ex-amines research monitoring the potential effects of GM crops and GM trees on target and non-target fungi.Parsing public databases for peer-reviewed publications about GM plant impacts on fungi yielded 149studies,a relatively modest number considering the diversity of crops and ecosystems studied.Analysis of these publications showed that the effects of GM plants expressing herbicide and insect tolerance on fungi are understudied while they dominate the GM area worldwide.Experiments monitoring the impact of GM crops and GM trees with enhanced antifungal activity towards target fungi showed,for the most part,significant decreases in disease severity caused by fungal pathogens.Significant changes,ex-pressed as an increase or decrease in fungal development,abundance,and diversity of non-target fungi,were observed in 18out of 60studies and all of them involved GM plants expressing traits that were unexpected to affect fungi.The re-maining 42studies did not identify a significant impact on fungal populations.Therefore,in spite of the fact that GM plants have been commercialized since 1996,no clear generalized trend can be identified and it appears that a case-by-case approach is the safest.
Key words:transgenic crops,transgenic trees,impact,target fungi,non-target fungi.
Re
′sume ′:Depuis deux de ′cennies,le ge ′nie ge ′ne ′tique permet de de ′velopper des essences agricoles et forestie `res ge ′ne ′tique-ment modifie
′es (GM)afin d’ame ′liorer les rendements et simplifier les pratiques culturales.Cela,associe ′au de ′ploiement de monocultures sur de grandes e
′tendues,peut ge ′ne ′rer un stress environnemental ainsi que des effets secondaires inde ′https://www.sodocs.net/doc/d26544954.html, commercialisation de plantes agricoles GM et le re ′cent de ′veloppement d’arbres GM suscitent des inquie ′tudes a `propos de leur impact potentiel sur l’environnement en ge
′ne ′ral et sur la biodiversite ′des organismes non cibles,en particu-lier.Les champignons sont pre
′sents partout sur la plane `te,occupent des fonctions cle ′s dans tous les e ′cosyste `mes,et sont intimement lie
′s aux ve ′ge ′taux depuis que ces derniers ont colonise ′les terres e ′merge ′es.Dans cette revue,les auteurs exami-nent les recherches portant sur les effets potentiels des essences agricoles et forestie
`res GM sur les champignons cibles et non https://www.sodocs.net/doc/d26544954.html, recherche dans les bases de donne
′es de publications re ′vise ′es par les pairs portant sur l’impact des essences agricoles et forestie
`res GM sur les champignons a permis de retrouver 149e ′tudes,un nombre relativement modeste consi-de
′rant la diversite ′des plantes agricoles et forestie `res et des e ′cosyste `mes e ′tudie ′s.L’analyse de ces publications montre que les effets des plantes GM tole
′rantes aux herbicides et aux insecticides sur les champignons sont sous-estime ′s,alors qu’el-les dominent les surfaces cultive
′es en plantes GM a `l’e ′chelle mondiale.Les e ′tudes portant sur l’impact des essences agri-coles et forestie
`res GM afin d’augmenter leur activite ′antifongique envers des agents pathoge `nes fongiques montrent,dans la plupart des cas,des diminutions significatives de la se
′ve ′rite ′des maladies.Des changements significatifs,exprime ′s sous forme d’augmentation ou de diminution du de
′veloppement,de l’abondance,ou de la diversite ′des champignons non cibles,ont e
′te ′observe ′s dans 18e ′tudes sur 60portant sur des plantes GM exprimant des caracte `res transge ′niques a priori sans ef-fet sur les champignons.Ainsi,bien que les plantes GM soient commercialise
′es depuis 1996,on ne peut pas identifier de tendance quand au risque qu’elles repre
′sentent pour les champignons et l’e ′tude au cas par cas semble l’approche la plus sure.
Mots-cle
′s :cultures transge ′niques,arbres transge ′niques,impact,champignons cibles,champignons non cibles.[Traduit par la Re
′daction]Received 5August 2010.Accepted 27October 2010.Published on the NRC Research Press Web site at er.nrc.ca on 20November 2010.F.Stefani.Universite
′Laval,Faculte ′de foresterie,de ge ′ographie et de ge ′omatique,Pavillon Abitibi-Price,2405,rue de la Terrasse,Que
′bec,QC G1V 0A6,Canada;Natural Resources Canada,Canadian Forest Service,Laurentian Forestry Centre,1055du P.E.P.S,P.O.Box 10380,Stn.Sainte-Foy,Que
′bec,QC G1V 4C7,Canada.R.Hamelin.1Natural Resources Canada,Canadian Forest Service,Laurentian Forestry Centre,1055du P.E.P.S,P.O.Box 10380,Stn.Sainte-Foy,Que
′bec,QC G1V 4C7,Canada.1Corresponding
author (e-mail:rhamelin@nrcan.gc.ca).
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1.Introduction
Intensive agricultural practices have resulted in field de-ployment of monocultures over large areas.The manage-ment of monocultures typically requires the use of chemical substances to control weeds and to limit insect damage and microbial diseases.These practices stress the development of plant varieties with new traits such as herbicide tolerance or increased resistance against potential attacks from viruses,bacteria,fungi or insects.For two decades,genetic engineer-ing has made it possible to develop some made-to-measure plants to facilitate the spreading of chemical pesticides and to improve yield.The surface planted with genetically modi-fied (GM)crops has been constantly increasing since 1996and reached 134million hectares in 2009,distributed over 25countries worldwide,and represented 8.7%of the total area of arable land (James 2009;FAO 2010).Soybean,cot-ton,maize,and canola are most often subjected to genetic transformation as 70%,48%,24%,and 20%,respectively,of the surface hosting these cultures have been planted with genetically engineered plants (James 2008).Herbicide toler-ance and insecticidal toxin production are the most common traits used in GM crops (Brookes and Barfoot 2009).
In forestry,the development of trees displaying desirable characteristics based on conventional breeding is difficult because of the long reproductive cycle of woody species.Genetic transformations and in vitro micro-propagation cir-cumvent these barriers and make it possible to easily and quickly develop trees with desirable traits from a large inter-specific pool of genes.Since the 1990s,progress has been made in tree transgenesis and the number of transformed woody species and field tests increased during the period 1990–2000(van Frankenhuyzen and Beardmore 2004).The most common woody species subjected to genetic transfor-mation belong to the genera Populus (47%),Pinus (19%),Eucalyptus (7%),Liquidambar (5%),and Picea (5%)(Marchadier and Sigaud 2005).The desired new traits are mainly of commercial interest such as improved vigour,al-teration of wood lignin content,abiotic stress (frost,dryness,salt)and herbicide tolerance,and increased resistance against insects or microbial pathogens (Tzfira et al.1998;Marchadier and Sigaud 2005;Teulieres and Marque 2007).However,most GM trees are still under development and field testing.There are only two instances of commercial cultivation of trees:(i )GM papaya,exhibiting resistance against ringspot virus,field deployed in the U.S.;and (ii )Bacillus thuringiensis (Bt)-transformed black poplars,field deployed in 2002on 200–300ha in China (www.gmo-safety.eu).
The commercial growing of genetically modified organ-isms (GMOs)in agriculture has raised public ecological awareness.The effects caused by transgene insertion and (or)expression raise the issue of the potential risks for the environment.The application of this technology to woody species has also given rise to intense public debate about en-vironmental risk issues (Mathews and Campbell 2000;Strauss et al.2009).The hazards associated with growing GM crops and GM trees are nearly the same.These include issues such as:vertical gene flow defined as contamination of wild plant genomes due to transgenic pollen,seeds and vegetative propagule dispersion;GM plant escape with po-
tential weedy behaviour;horizontal gene transfer;and un-foreseeable effects of the insertion and (or)expression of the transgene on non-target organisms.Nevertheless,tempo-ral and spatial scales of the interactions of GM trees with their environment differ from transgenic crops (van Frank-enhuyzen and Beardmore 2004).Trees are long-lived peren-nials and they develop several biotic interactions with soil microbial communities for a longer period than crops.Therefore,one can consider that the risk of GM trees to non-target organisms is potentially more important because of the longer exposure of microbial communities to the re-combinant organisms as compared with GM crops.
The GMOs impact on non-target organisms typically in-volves plants that are genetically engineered to increase their resistance against insects and microbial pathogens.Plants mainly interact with their environment through their root system.Soil microorganisms are potentially more ex-posed to the new traits expressed by GM plants,among which fungi are likely the most important.The main func-tions of soil fungi are as decomposition and providers of mutualistic benefits (Christensen 1989;Bridge and Spooner 2001).Along with fungal pathogens and parasites,fungi are involved in shaping plant community structure and dynam-ics.Among soil fungi,mycorrhizae are the fungal group dis-playing the closest relationships with their hosts.They play key roles in the primary biogeochemical processes (Read and Perez-Moreno 2003;Leake et al.2004;Read et al.2004)and are thereby deeply involved in soil fertility.Most terrestrial plants in temperate,boreal,tropical,and subtropi-cal regions are colonized by mycorrhizal fungi.
The innocuousness of GMOs toward non-target organisms is one of the five points listed by the Plant Biosafety Office (PBO)of the Canadian Food Inspection Agency (CFIA)that must be satisfied prior to the release of transformed plants (Finstad et al.2007).Here,we survey 20years of research monitoring desirable and undesirable impacts of GM crops and GM trees on targeted fungi (fungal pathogens)and non-targeted fungi (mostly mycorrhizal fungi).
The ISI Web of Science online database was parsed by targeting the following key words in publication titles:ge-netically engineered,genetically modified,transgenic,and transformed.These key words were associated in publication titles with the following plant key words:alfalfa,barley,canola,carnation,cotton,maize,papaya,petunia,potato,rice,sweet pepper,soybean,squash,sugar beet,tobacco,to-mato,wheat,apple,aspen,birch,eucalyptus,liquidambar,pine,poplar,and spruce.Database parsing was repeated with the previous plant key words in their plural form and finally with the corresponding genus name.Moreover,the database was parsed for articles that included in their title the words glyphosate,herbicide,and insect,associated either with the words tolerant or resistant,plus the words fungi,fungus,or fungal included in all the fields.Over 6700publi-cations were retrieved.Additional searches were performed by parsing the Google Scholar search engine and PubMed and JStor databases with different combinations of the fol-lowing key words:GMOs,GM trees,GM crops,impact,tar-get fungi,non-target fungi,fungus,and fungal.Database parsing was concluded in April 2010.The software Papers v1.9.3(https://www.sodocs.net/doc/d26544954.html,)was used to parse databases,store,organize,and analyze publications.
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2.Interaction of genetically modified crops with fungi
2.1Overview
From 1991to 2010,we recorded 117studies assessing the potential effect of transgenic crops on fungi (Table 1).In-vestigations on the effect of GM plants on fungi were per-formed mainly under controlled conditions (in 96studies out of 117).The analysis of these publications showed that the potential impact of transgenic plants on target fungi has been better monitored than the impact on non-target fungi.During this period,we recorded 84studies examining GM crop impacts on target fungi versus 35studies towards non-target fungi.Figure 1A shows the progress in field planting GM crops with the number of impact studies on target and non-target fungi.While the average area planted with GM crops continually increased by 10million ha per year since 2000,the number of surveys of GM crop impacts on fungi remained steady with 10studies per year on average,includ-ing the year 2008when 17articles were published.From 1996to 2009,studies on non-target fungi represented ap-proximately 25%of the annual articles published each year about GM crop relationships with fungi.The assessment of GM plant impacts on target and non-target fungi (Fig.2A)mainly involved plants from genera Nicotiana (23%),Oryza (19%),and Triticum (16%).As might be expected,69%of the new traits expressed in GM plants tested against target and non-target fungi were related to fungal disease resist-ance (Fig.2B).Taking into consideration only studies mon-itoring GM crops on non-target fungi,this trait represented 13%,while insect resistance was the trait that was most fre-quently monitored (40%,data not shown).This is not sur-prising as Bt crops have been spread over 200million ha since 1996(James 2009)and have virtually replaced tradi-tional cultivars in areas like the U.S.,China,India,and South Africa (Jongsma et al.2010).The potential effects of GM crops resistant to fungal pathogens on non-target fungi do not seem to be a topical issue in the field of the environ-mental risk assessment of GM plants.Similarly,studies monitoring the impact of herbicide tolerant plants on fungi represented 4%of the studies retrieved (Fig.2B).This was unexpected as herbicide tolerance is the dominant trait in commercialized transgenic cultures,representing 63%of the surface cultivated with GM crops in 2008(James 2008).2.2Genetically modified crop impacts on target fungi GM plants with enhanced antifungal activities have been developed and tested against fungal pathogens belonging mainly to the genera Rhizoctonia (22%),Magnaporthe (15%),Fusarium (11%),Botrytis (6%),Sclerotinia (6%)and Erysiphe (5%,Fig.3A).There were 139assays that showed the efficiency of GM plants to limit fungal pathogen establishment and development.Most of them were trans-formed with vectors carrying pathogenesis-related (PR)pro-teins like b -1,3-glucanase (PR-2),chitinase (PR-3),thaumatine like protein (PR-5)under the control of cauli-flower mosaic virus 35S or ubiquitin promoters.The mean exposure time of target fungi to transgenic plants was 28days (median =13,min =1,max =473)and symptom severity based on counting and measuring necrotic lesions was the most common response variable recorded.
Among the few studies that tested GM crop resistance against target fungi in the field,six surveys out of seven in-volved transgenic wheat.The GM plants with increased tol-erance to fungal pathogens under controlled conditions did not necessarily display the same level of resistance when field deployed.For example,Anand et al.(2003)showed that a wheat line expressing the thaumatin-like protein (tlp)transgene was significantly less susceptible to scab (Fusa-rium graminearum )when grown in a greenhouse (reduced mean number of infected spikelets per head).However,dis-ease severity observed on this transgenic line during field tests was either similar to or higher than the susceptible con-trols.They hypothesized that lesion-mimic phenotypes could compromise the plant’s ability to undergo continuous patho-gen pressure in the field.Shin et al.(2008)observed dispa-rate results between greenhouse-and field-grown transgenic wheat carrying a barley chitinase transgene.Chitinase-ex-pression levels were similar between controls and transgenic lines albeit these last ones had enhanced resistance to scab in the greenhouse but similar susceptibility to controls in the field.Other studies that evaluated,qualitatively or quan-titatively,GM plant performance in the field showed im-proved fungal resistance with respect to controls (Cober et al.2003;Schlaich et al.2006;Zhao et al.2006;Mackintosh et al.2007;Luo et al.2008).
2.3Genetically modified crop impacts on non-target fungi
2.3.1No evidence of negative effects on non-target fungi The issue of the potential effects of GMO cultures on non-target fungi has been addressed either by monitoring the level of colonization and development of vesicular ar-buscular mycorrhizal fungi (mainly represented by Glomus mosseae and Glomus intraradices )or by investigating soil fungal communities by means of fungal culturing,bio-markers or molecular tools (Fig.3B).Of 56assays,42showed no effect on non-target fungi associated with GM plants or fungi occurring in their vicinity.The mean expo-sure time of non-target fungi to transgenic plants was 63days (median =38,min =12,max =473).Of 35studies on non-target fungi,13were performed in the field.Only two studies evaluated the impact on both target and non-target fungi.Vierheilig et al.(1993)showed Nicotiana sylvestris genetically transformed with different chitinase genes to be more resistant to Rhizoctonia solani while the colonization by the endomycorrhizal fungus G.mosseae was not affected.It was proposed that proteins or alkalisoluble polysacchar-ides might cover the chitin layer of the fungal cell wall and prevent chitinase from binding to endomycorrhizal hyphae.Under controlled conditions,Turrini et al.(2004a )trans-formed aubergine plants to constitutively express the Dm-AMP1antimicrobial defensin protein and monitored the po-tential effects on the soilborne fungal pathogen Verticillium albo-atrum ,on the phytopathogenic fungus leaf infecting Botrytis cinerea ,and on the endomycorrhizal G.mosseae .They showed that the growth of V.albo-atrum colonies was reduced by 49%to 71%with respect to controls by the re-lease of active antifungal protein from the roots of trans-formed aubergine plants.The necrotic areas of leaves infected with B.cinerea were also reduced by 36%to
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100%.On the other hand,host recognition responses and es-tablishment of the endomycorrhizal symbiosis was not af-fected in these transgenic lines.To explain the differential effect of Dm-AMP1-transformed aubergines on the benefi-cial endomycorrhizal symbiosis,the authors hypothesized that G.mosseae hyphal membranes would not expose at their surface patches containing sphingolipids to which Dm-AMP1binds and then causes damage to membranes (The-vissen et al.2000,2003).
Three other studies assessed the potential effects of GM crops with enhanced fungal resistance towards non-target fungi.Vierheilig et al.(1995)inoculated G.mosseae
to
Fig.1.Increase in the global hectarage of biotech crops in the period 1996–2009(red line).Stack histograms show the number of studies investigating the potential impact of (A)genetically modified (GM)crops (n =110);and (B)GM trees (n =31)on target fungi (dark grey)and on non-target fungi (light grey)in the period
1996–2009.
Fig.2.(A)Genera of genetically modified (GM)crops investigated for their potential impact on fungi (n =164)and (B)new traits ex-pressed in the GM crops monitored,tested against target and non-target fungi (n =164).
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tobacco constitutively expressing pathogenesis-related pro-teins like chitinase or glucanase and observed no difference in either the final level of root colonization or during the time course of colonization between control and transformed tobacco.The roots of defensin-transformed aubergine were as susceptible as those of non-transformed aubergine to colonization by G.mosseae (Turrini et al.2004a ,2004b ).Girlanda et al.(2008)investigated the potential impact on endomycorrhizal fungi and saprotrophic fungi associated with the rhizosphere and phyllosphere of tomatoes geneti-cally engineered to express chitinase and glucanase proteins.They found no significant difference in the frequency and intensity of endomycorrhization and in the arbuscular qual-ity between transgenic tomatoes and controls,after 2and 8months of interaction.Furthermore,based on the isolation and identification of over 20500fungal colonies retrieved in the rhyzosphere and phyllosphere of transgenic tomatoes and controls,no evidence of change within the two fungal communities was observed,after the same time of interac-tion with transgenic tomatoes.
2.3.2Genetically modified plants galvanize fungi:unexpected effects
Until now,techniques used to transform plants have not made it possible to control either the number or the position of the transgene(s)incorporated within the recipient plant genome.This sometimes leads to the expression of unex-pected traits (pleiotropic effects)that are not directly related
to transgene expression.Major changes can be observed when pleiotropic effects result in modifying plant root exu-dation.Indeed,root exudates play a key role in structuring soil microbial communities (Kowalchuk et al.2003;Bais et al.2006;Broeckling et al.2008).The most acute conse-quence recorded in the literature due to pleiotropic effect was observed in transgenic insect-resistant lines of cotton.In China,64varieties of pest-resistant cotton were grown on 3.7million hectares representing 70%of the total culti-vated cotton culture area (Stone 2008).Since their field de-ployment,it has been observed that certain GM cotton lines were as susceptible to Fusarium and Verticillium wilts as cultivars lacking resistance.Li et al.(2009b )showed that root exudates of Cry1Ac/CpTI-and Cry1Ac-transformed lines contained more sugar (fructose,maltose,and an un-known sugar)than parental control lines.This augmentation of sugar concentration in root exudates favoured the devel-opment of the fungal pathogen Fusarium oxysporum within the rhizosphere,increasing cotton plant mortality.Despite the fact that these GM plants circumvent mortality due to Lepidoptera attacks and avert the use of 650000tons of in-secticides,fungicide use is now required to control Fusa-rium and Verticillium (Stone 2008;Li et al.2009b ).Another instance of unexpected increased fungal activity is seen in transgenic wheat expressing both chitinase and glu-canase.Bieri et al.(2003)showed that some transgenic wheat lines were more susceptible to Blumeria graminis f.sp.tritici while other transgenic lines displayed a resistant phenotype in a leaf bioassay.They also observed that plants displaying the highest level of these two anti-fungal en-zymes within their tissues were the most susceptible to the powdery mildew.The authors suspected the high level of b -1,3-glucanase to be responsible for the decrease in plant resistance against B.graminis .They hypothesized that glu-canase could interfere with papillae formation during the in-fection stage as they are made of callose (Aist 1976),which is a substrate of b -1,3-glucanase.Kremer et al.(2005)ob-served that the interaction between glyphosate treatment and glyphosate-resistant (GR)soybean significantly in-creased the biomass of three out of four Fusarium strains in root exudates of GR soybean compared with non-GR culti-vars treated with glyphosate.The authors showed that the root exudates of GR soybean released higher carbohydrate and amino acid contents than non-GR cultivars.Moreover,glyphosate treatment also enhanced these compounds in root exudates in both GR and non-GR cultivars.
Five other studies demonstrated that endomycorrhizal fungi and other soil fungi were stimulated when associated with GM plants.Donegan et al.(1995)observed a transient increase in the colony-forming units (CFU)of cultivable fungi from soil samples associated with two lines of insect-tolerant cotton.Staehelin et al.(2001)showed that the gene enod40,involved in formation of root nodules,upregulates mycorrhizal formation and development.The overexpression of enod40in alfalfa significantly increased the frequency of
arbuscules and vesicles in roots.He
′nault et al.(2006)inves-tigated the potential impact of lignin-modified tobacco resi-dues on soil fungal community.By measuring the proportion of double unsaturated chain fatty acids,indicative of the presence of fungi,they observed that the soil fungal com-munity incubated for 14days with residues from
one
Fig.3.(A)Fungal genera targeted by GM crops (n =158)and (B)genera of the non-targeted fungi monitored (n =56).
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tobacco line was significantly increased compared with con-trols.O’Callaghan et al.(2008)monitored fungal commun-ities associated with three lines of potato genetically modified to express the antimicrobial peptide magainin,by counting the number of fungal CFUs on the surface of leaves,roots,and tubers,after 2and 4months of field growth.Results showed contrasting effects at time of har-vest.The number of fungal CFUs was significantly higher in roots and lower in tubers of the transgenic line D9,with respect to unmodified parental lines and unrelated cultivars.The two other transformed lines did not show any difference from the controls.Contrasting results were also observed in the abundance of saprophytic fungi isolated from the rhizo-sphere of maize expressing the Cry1Ab protein (Oliveira et al.2008).Thirty days after sowing,the fungal CFUs were significantly more abundant in soil samples from one trans-formed line while significantly less abundant in soil samples from another transformed line.
Finally,Weinert et al.(2009)analyzed the soil fungal di-versity during three developmental stages of two GM potato lines genetically modified to accumulate the carotenoid zeaxanthin in their tubers.They observed a significant shift in fungal denaturing gradient gel electrophoresis (DGGE)fin-gerprints based on the internal transcribed spacer (ITS)se-quences between GM potato lines and their parental cultivars.The differences observed between the soil fungal communities associated with four commercial potato cultivars were higher than those observed between the two transgenic potato lines and their parental counterparts.Hence,in this case,the genetic modification of potatoes had less impact on the soil fungal community than the introduction of different potato cultivars.Therefore,the effects of transgenic crops on soil microorgan-isms would have to be interpreted in light of the effects stressed by shifting cultivars and other natural variability.2.3.3Genetically modified plants with negative effects on non-target fungi
Five assessments of GM impact on non-target fungi re-corded deleterious effects (Staehelin et al.2001;Turrini et al.2004b ;Castaldini et al.2005;O’Callaghan et al.2008;Oliveira et al.2008).None of these transgenic plants were transformed to express anti-fungal proteins.Staehelin et al.(2001)observed that the downregulation of the enod40tran-scription within transgenic lines of Medicago truncatula re-sulted in a significantly lower colonization of roots by G.intraradices compared with controls.In three cases out of five,these assessments involved plants genetically engi-neered to increase their insect tolerance through the expres-sion of the insecticidal toxin encoded by the Cry1Ab gene from Bacillus thuringiensis .Turrini et al.(2004b )compared the effect of root exudates of the lines Bt corn 176and Bt corn 11on G.mosseae pre-symbiotic growth and hyphal dif-ferential morphogenesis.The line Bt corn 176significantly reduced the length of G.mosseae mycelium and the number of appressoria developing infection units as 35.7%of ap-pressoria were not able to colonize roots,35days after inoc-ulation.A higher level of Cry1Ab toxin was measured in the line Bt corn 176(80.63Cry1Ab /g protein)that negatively affected G.mosseae compared with the line Bt corn 11(<0.55Cry1Ab /g protein)that was shown to be not delete-rious on the endomycorrhizal symbiosis.Castaldini et al.
(2005)monitored the development of endomycorrhizal in-fection units and the endomycorrhizal colonization of the lines Bt corn 176and Bt corn 11.In both transformed lines,the intraradical colonization by G.mosseae was significantly lower (about 50%)compared with wild type,after 8and 10weeks of interaction under controlled conditions.The num-ber of entry points developing arbuscules at 8days was sig-nificantly reduced in the roots of the lines Bt corn 11and Bt corn 176by 72%and 67%,respectively.The percentage of root length colonized by arbuscular mycorrhizal fungi was significantly lower in Medicago sativa grown for four months in soil containing Bt corn 11line residues.The rea-sons for which Bt corn lines are less susceptible to endomy-corrhizal colonization remain unknown.
3.Interaction of genetically modified trees with fungi
3.1Overview
The potential impact of transgenic trees on fungi has been assessed in 32studies published between 1996and 2010(Table 2).Since 2000,on average three studies monitoring the potential impact of GM trees on fungi have been pub-lished each year.This is one-third the number of studies re-lated to GM crop impact on fungi during the same interval.The potential impact of GM plants on non-target fungi has prompted more attention in forestry as they accounted for 50%of the studies recorded compared with 25%in agricul-ture (Figs.1A and 1B),hence this is an area of research that deserves increased attention.Valenzuela et al.(2006)showed that the number of experiments field testing GM trees worldwide increased from 7to 36in the period 1996–2001.The number of outdoor plots recorded in the USA and Europe was 46in 2003,354in 2005(Robischon 2006)and it reached 528in 2010(www.gmo-safety.eu).Poplar,pine,apple and eucalyptus were the dominant species transformed and field deployed.The three principal traits expressed in these outdoor experiments were disease resistance (20%),selectable marker gene (16%),and herbicide tolerance (14%)(Fig.4A).Fungal disease resistance was the most fre-quent trait (66%)expressed by GM trees that were studied regarding their potential impact on fungi,followed at a dis-tance by altered lignin composition (12%)(Fig.4B).These transgenic trees belong for the most part to the genera Pop-ulus (35%),Malus (29%),Betula (21%)and Picea (8%)(Fig.5).No data about the potential impact of GM eucalyp-tus and GM pine on fungi were retrieved.
Fungal disease resistance was mainly increased within GM trees by the insertion and expression of genes encoding for chitinolytic enzymes.Chitinases have been isolated from bacteria,fungi and plants and can be classified in two groups according to the way they cleave chitin.Endochiti-nase degrades chitin wall (a major component of the fungal cell wall)by randomly cleaving internal sites of the chitin molecule whereas exochitinase attacks the chitin molecule from its nonreducing end (Cohen-Kupiec and Chet 1998).The insertion of gene(s)encoding chitinase(s)within a plant genome is thought to promote the fungal disease resistance of this plant via increased chitinolytic activity.
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