Bacterial Endophytes and Their Interactions with Hosts


Bacterial Endophytes and Their Interactions with Hosts

Mónica Rosenblueth and Esperanza Martínez-Romero

 Centro de Ciencias Genómicas, Universidad Nacional Autóma de México, Apdo. Postal 565-A, Cuernavaca, México

Submitted 9 September 2005. Accepted 8 March 2006.

Recent molecular studies on endophytic bacterial diversity have revealed a large richness of species. Endophytes pro- mote plant growth and yield, suppress pathogens, may help to remove contaminants, solubilize phosphate, or contribute assimilable nitrogen to plants. Some endophytes are seed- borne, but others have  mechanisms to colonize the plants that are being studied. Bacterial mutants unable to produce secreted proteins are impaired in the colonization process. Plant genes expressed in the presence of endophytes provide clues as to the effects of endophytes in plants.  Molecular analysis showed that plant defense responses limit bacterial populations inside plants. Some human pathogens, such as Salmonella spp., have been found as endophytes, and these bacteria are not removed by  disinfection procedures that eliminate superficially occurring bacteria. Delivery of endo- phytes to the  environment or agricultural fields should be carefully evaluated to avoid introducing pathogens.

 

Additional keywords:  nitrogen fixation,  pathogenic  bacteria, plant bacteria, plant colonization.

 

 

Plants and animals normally associate with diverse microor- ganisms. In the gut, bacteria have a remarkable role to stimu- late immunity and development (Hooper et al. 2001). Similarly plant  bacteria  stimulate  plant  defense  responses  (de  Matos Nogueira et al. 2001). Bacteria on roots and in the rhizosphere benefit from root exudates,  but  some bacteria and fungi are capable of entering the plant as endophytes that do not cause harm and could establish a mutualistic association (Azevedo et al. 2000; Hallmann et al. 1997; Perotti 1926). Plants constitute vast and diverse niches for endophytic organisms. Endophytic bacteria have been isolated from a large diversity of plants as reviewed by Sturz and associates (2000). Plants reported to har- bor endophytes are shown in Table 1, but most likely, there is not a single plant species devoid of endophytes. The few exam- ples of apparent absence of internal populations may be because some microorganisms are not easily isolated or cultured.

In  general endophytic bacteria occur at lower  population densities  than  rhizospheric  bacteria  or  bacterial  pathogens (Hallmann  et  al.  1997;  Rosenblueth  and  Martínez-Romero

2004). It has not been resolved whether plants benefit  more from an endophyte than from a rhizospheric bacterium or if it is more advantageous for bacteria to become endophytic com- pared with rhizospheric. It is still not always clear which popu- lation of microorganisms (endophytes or rhizospheric bacteria) promotes  plant  growth;  nevertheless,  benefits  conferred  by endophytes are well recognized and will be presented here.

 

Corresponding author: Esperanza Martínez-Romero; E-mail: emartine@ccg.unam.mx

 

Endophytic populations, like rhizospheric populations,  are conditioned by biotic and abiotic factors (Fuentes Ramírez et al. 1999; Hallmann et al. 1997, 1999; Seghers et al. 2004), but endophytic bacteria could be better protected from biotic and abiotic  stresses than  rhizospheric  bacteria  (Hallmann  et  al.

1997).

Endophytic bacteria in a single plant host are not restricted to a single species but comprise several genera and species. No one knows if communities inside plants  interact, and it has been speculated that beneficial effects are the combined effect of their activities. In this review, we will first address the di- versity of endophytes. Unfortunately, some of the older papers describing bacteria inside plants have used methods that do not allow an accurate classification of endophytic bacteria.

Criteria to recognize “true” endophytic bacteria have  been published (Reinhold-Hurek and Hurek 1998a) and this requires not only the isolation from surface-disinfected tissues but also microscopic  evidence  to  visualize  “tagged”  bacteria  inside plant tissues. The latter criterion is not always fulfilled. Use of the term putative endophytes has been recommended for those not validated  microscopically. True endophytes may also be recognized by their capacity to reinfect disinfected seedlings.

Endophytic bacteria have been studied mainly after culturing in laboratory media, but a more complete scheme is emerging, using methods that do not require the bacteria to be cultured and that make use of the analysis of sequences from bacterial genes obtained from DNA isolated from inside plant tissues (Chelius and Triplett 2000a; Engelhard et al. 2000; Miyamoto et al. 2004; Reiter et al. 2003; Sessitsch et al. 2002b). A fol- lowing molecular approach studying wheat endophytes in Aus- tralia  revealed  a  larger diversity of actinobacteria than  that obtained by culturing endophytes (Conn and Franco  2004). Evidence that there are endophytic bacteria that have not yet been cultured also comes from the study of citrus endophytes by  denaturing  gradient  gel  electrophoresis  profiles  of  16S rRNA gene fragments amplified from total plant DNA. Some bands did not match any of the isolated bacteria grown in cul- ture media  (Araujo et al. 2002). In contrast, no differences were  obtained  by culturing or  culture-independent methods and both revealed similar bacteria from the genera Pseudomo- nas and Rahnella in Norway spruce seeds (Cankar et al. 2005).

 

Diversity and populations of microorganisms recovered as endophytes.

Mycorrhizal fungi are ancient plant partners (Simon et  al.

1993) widespread among plants. Mycorrhiza benefits to plants are well known but agricultural applications in the field have not frequently led to substantial increases in crop yields. Some trees are incapable of growing without their  mycorrhiza, and mycorrhizal fungi are fully dependant on the plant for growth. Recently, host specificity has begun  to be recognized, using

 

 

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molecular analysis based on the sequence of ribosomal genes (Chelius and Triplett 1999; Jacquot et al. 2000;  Kjoller and Rosendahl 2000) and other genes, such as  those for actin or elongation factors (Helgason et al. 2003). Interestingly, some mycorrhizal  fungi  themselves  have  endosymbiotic  bacteria (Glomeribacter gigasporarum; Jargeat et al. 2004). Like my-

 

corrhiza,  other  endophytic fungi  completely depend on  the plant and its inside conditions for growth. Some  endophytic fungi  have  been  shown  to  protect  plants  from  herbivores (Schardl et al. 2004) or to be responsible for the synthesis of novel and useful secondary products (Strobel et al. 2004). Rain forest destruction may lead not only to the loss of valuable tree

 

 

 

Table 1. Examples of reported bacterial endophytes and plants harboring them

 

Endophytes                                                                    Plant species                                                             Reference

α Proteobacteria

Azorhizobium caulinodans                         Rice                                                      Engelhard et al. 2000

Azospirillum brasilense                               Banana                                                  Weber et al. 1999

Azospirillum amazonense                           Banana, pineapple                                 Weber et al. 1999

Bradyrhizobium japonicum                        Rice                                                      Chantreuil et al. 2000

Gluconacetobacter diazotrophicus             Sugarcane, coffee                                  Cavalcante and Döbereiner 1988; Jiménez-Salgado et al. 1997

Methylobacterium mesophilicuma                       Citrus plants                                          Araujo et al. 2002

Methylobacterium extorquens                     Scots pine, citrus plants                         Araujo et al. 2002; Pirttilä et al. 2004

Rhizobium leguminosarum                         Rice                                                      Yanni et al. 1997

Rhizobium (Agrobacterium) radiobacter    Carrot, rice                                              Surette et al. 2003

Sinorhizobium meliloti                               Sweet potato                                         Reiter et al. 2003

Sphingomonas paucimobilisa                                Rice                                                      Engelhard et al. 2000

β Proteobacteria

Azoarcus sp.                                               Kallar grass, rice                                    Engelhard et al. 2000; Reinhold-Hurek et al. 1993

Burkholderia pickettiia                                                Maize                                                    McInroy and Kloepper 1995

Burkholderia cepaciab                                               Yellow lupine, citrus plants                   Araujo et al. 2001; Barac et al. 2004

Burkholderia sp.                                        Banana, pineapple, rice                         Weber et al. 1999; Engelhard et al. 2000

Chromobacterium violaceuma                               Rice                                                      Phillips et al. 2000

Herbaspirillum seropedicae                        Sugarcane, rice, maize, sorghum, banana    Olivares et al. 1996; Weber et al. 1999

Herbaspirillum rubrisulbalbicans               Sugarcane                                             Olivares et al. 1996

γ Proteobacteria

Citrobacter sp.                                           Banana                                                  Martínez et al. 2003

Enterobacter spp.                                       Maize                                                    McInroy and Kloepper 1995

Enterobacter sakazakiia                                             Soybean                                                Kuklinsky-Sobral et al. 2004

Enterobacter cloacaea                                                Citrus plants, maize                               Araujo et al. 2002; Hinton et al. 1995

Enterobacter agglomeransa                                    Soybean                                                Kuklinsky-Sobral et al. 2004

Enterobacter asburiae                                Sweet potato                                         Asis and Adachi 2003

Erwinia sp.                                                 Soybean                                                Kuklinsky-Sobral et al. 2004

Escherichia colib                                                            Lettuce                                                  Ingham et al. 2005

Klebsiella sp.                                              Wheat, sweet potato, rice                      Engelhard et al. 2000; Iniguez et al. 2004; Reiter et al. 2003

Klebsiella pneumoniaeb                                             Soybean                                                Kuklinsky-Sobral et al. 2004

Klebsiella variicolab                                                     Banana, rice, maize, sugarcane              Rosenblueth et al. 2004.

Klebsiella terrigenaa                                                     Carrot                                                   Surette et al. 2003

Klebsiella oxytocab                                                       Soybean                                                Kuklinsky-Sobral et al. 2004

Pantoea sp.                                                Rice, soybean                                        Kuklinsky-Sobral et al. 2004; Verma et al. 2004

Pantoea agglomerans                                Citrus plants, sweet potato                     Araujo et al. 2001, 2002; Asis and Adachi 2003

Pseudomonas chlororaphis                        Marigold (Tagetes spp.), carrot             Sturz and Kimpinski 2004; Surette et al. 2003

Pseudomonas putidaa                                                 Carrot                                                   Surette et al. 2003

Pseudomonas fluorescens                           Carrot                                                   Surette et al. 2003

Pseudomonas citronellolis                          Soybean                                                Kuklinsky-Sobral et al. 2004

Pseudomonas synxantha                            Scots pine                                             Prittilä et al. 2004

Salmonella entericab                                                   Alfalfa, carrot, radish, tomato               Cooley et al. 2003; Guo et al. 2002; Islam et al. 2004

Serratia sp.                                                Rice                                                      Sandhiya et al. 2005

Serratia marcescensa                                                    Rice                                                      Gyaneshwar et al. 2001

 

Stenotrophomonasa                                                      Dune grasses (Ammophila arenaria and

Elymus mollis)

 

Dalton et al. 2004

 

Firmicutes

Bacillus spp.                                              Citrus plants                                          Araujo et al. 2001, 2002

Bacillus megaterium                                   Maize, carrot, citrus plants                    Araujo et al. 2001; McInroy and Kloepper 1995; Surette et al. 2003

Clostridium                                                Grass Miscanthus sinensis                      Miyamoto et al. 2004

Paenibacillus odorifer                                Sweet potato                                         Reiter et al. 2003

Staphylococcus saprophyticusb                            Carrot,                                                  Surette et al. 2003

Bacteroidetes

Sphingobacterium sp.a                                               Rice                                                      Phillips et al. 2000

Actinobacteria

Arthrobacter globiformis                            Maize                                                    Chelius and Triplett 2000a

Curtobacterium flaccumfaciens                  Citrus plants                                          Araujo et al. 2002

Kocuria varians                                         Marigold                                               Sturz and Kimpinski 2004

Microbacterium esteraromaticum               Marigold                                               Sturz and Kimpinski 2004

Microbacterium testaceum                          Maize                                                    Zinniel et al. 2002

Mycobacterium sp.b                                                      Wheat, Scots pine                                 Conn and Franco 2004; Prittilä et al. 2005

Nocardia sp.b                                                                    Citrus plants                                          Araujo et al. 2002

Streptomyces                                              Wheat                                                   Coombs and Franco 2003a

a   Opportunistic human pathogenic bacteria.

b   Common human pathogenic bacteria.

 

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species  but  also  of  unknown  endophytes,  especially  fungi (Strobel et al. 2004). Fungal endophytic interactions  will not be further reviewed here, as excellent reviews  have been re- cently published on this topic (Hause and Fester 2005; Schardl et al. 2004; Schulz and Boyle 2005; Strobel and Daisy 2003; Strobel et al. 2004).

Methods for the isolation of bacterial endophytes have been reviewed extensively (Hallmann et al. 1997;  Reinhold-Hurek and Hurek 1998a). The use of sodium hypochlorite for disin- fecting plant surfaces is common. Residual sodium hypochlo- rite may affect growth or induce mutagenesis and death of mi- croorganisms, thus  making it necessary to rinse the tissues with sodium thiosulfate to remove all the residual sodium hy- pochlorite (Miché and Balandreau 2001), although this is not a common practice.

A review of published endophytic bacteria was reported by Hallmann and associates in 1997, but the list is no longer com- plete, as there is much interest in this area and new endophytes are continuously being reported (Table 1).

Remarkably, Salmonella strains have been detected as endo- phytes  in  alfalfa  sprouts.  Outbreaks  with  these  bacteria  in alfalfa sprouts have been recorded in North America, Asia and Europe since 1995 (Ponka et al. 1995). It has been proposed that alfalfa plants and seeds be colonized with safe bacteria to out-compete human pathogens. For example, Enterobacter ab- suriae  was  found to eliminate Salmonella enterica and the enterohemorrhagic Escherichia coli from Arabidopsis thaliana seeds (Cooley et al. 2003). It is worrisome that there may be human or opportunistic pathogens among plant endophytes.

It  seems  that  the  bacteria  best  adapted  for  living  inside plants are naturally selected. Endophytes are recruited out of a large pool of soil or rhizospheric species and clones. Indeed, in a large study conducted on potato-associated bacterial commu- nities, species richness  and diversity was lower for fungal- antagonistic bacteria  inside roots than in the rhizosphere of potato  (Berg  et  al.  2005a). Germida and  associates  (1998) found that the endophytic population was less diverse than the root-surface population and the endophytes appeared to origi- nate from the latter. Mavingui and associates (1992) found that there are different populations of Bacillus  polymyxa in soil, rhizosphere, and rhizoplane and that  wheat roots select spe- cific populations. Rosenblueth  and Martínez-Romero (2004) found, both by multilocus enzyme electrophoresis and by plas- mid  patterns, that  Rhizobium etli  strains that  were isolated from inside maize stems were selected subsets of the total pool of  Rhizobium etli found in rhizosphere, roots, or Phaseolus vulgaris nodules. Rhizobium etli is found as a natural  endo- phyte of maize plants in traditional agricultural fields in which maize and bean are grown in association  (Gutiérrez-Zamora and Martínez-Romero 2001). In planta and ex planta popula- tions of Pseudomonas species could be differentiated by bio- chemical characteristics (van Peer et al. 1990).

Competition experiments with endophytes have shown that some endophytes are more aggressive colonizers and displace others. This  was  observed  with  Pantoea  sp.  out-competing Ochrobactrum sp. in rice (Verma et al. 2004) and with differ- ent   Rhizobium   etli   strains   in   maize   (Rosenblueth   and Martínez-Romero 2004).  However, when the host range of a large diversity of endophytes was analyzed, a seeming lack of strict specificity was observed (Zinniel et al. 2002).

The presence of different endophytic species in soybean de- pended on the plant genotype, the plant age, the  tissue sam- pled, and also on the season of isolation (Kuklinsky-Sobral et al. 2004). The soil type determined to a large extent the endo- phytic population in wheat (Conn and Franco 2004). Correla- tions  to growth promotion of  tomato plants were observed with inocula levels that promoted endophytic populations but

 

not rhizospheric populations (Pillay and Nowak 1997). On the other hand, the genus Azospirillum, one of the best-character- ized  plant  growth-promoting  bacteria,   exerts  its  benefits mainly in the rhizosphere (Somers et al. 2004) and rarely colo- nizes the plant inner cortical tissues  (Schloter and Hartmann

1998; Weber et al. 1999).

The  addition  of  the  herbicide  glyphosphate  produced  a modification of the endophytic composition of soybean plants (Kuklinsky-Sobral et al. 2005). Similar results were  obtained by  inoculating a  genetically modified  Enterobacter cloacae strain in citrus seedling (Andreote et al. 2004). The analysis by genomic fingerprinting of the diversity of Bacillus pumilus and Pantoea agglomerans isolated from surface-disinfected leaves showed that populations inside citrus do not seem to be clones derived from a single genotype (Araujo et al. 2001).

The population density of endophytes is highly variable, de- pending mainly on the bacterial species and host genotypes but also in the host developmental stage, inoculum density, and en- vironmental conditions (Pillay  and Nowak 1997; Tan et al.

2003). Interestingly, this is also the case with epiphytic  (on leaf surface) bacteria, which are highly variable in  number, varying around 1,000-fold the population size of one individ- ual bacterial species from leaf to leaf. Total bacterial popula- tion  sizes  on  inoculated  leaves  varied   by  about  30-fold (Mercier and Lindow 2000).

Clostridia were detected in surface-disinfected grass leaves, stems, and roots. A group of clostridia was found exclusively in one of the grass species analyzed and not in the surrounding soil (Miyamoto et al. 2004). Some endophytes are very scarce or absent in soil (Reinhold-Hurek and Hurek 1998a).

Endophytic  N2-fixing  bacteria  seem  to  constitute  only  a small proportion of total endophytic bacteria (Barraquio et al.

1997; Ladha et al. 1983; Martínez et al. 2003), and increasing N2-fixing populations in plants has been considered as a possi- bility  to increase nitrogen fixation.  Nitrogen-fixing bacteria were identified in sweet potato in N-poor soils with an analysis that consisted of amplifying nitrogenase (nifH) genes by poly- merase chain reaction  (Reiter et al. 2003). The resulting se- quences, presumably derived from endophytes, resembled those from rhizobia, including Sinorhizobium meliloti, Sinorhizobium sp. strain NGR234, and Rhizobium etli. Other detected bacteria were Klebsiella spp. and Paenibacillus odorifer  (Reiter et al.

2003). It is interesting that, in this case and perhaps in relation to the methodology used, a dominance of  rhizobia was ob- served, accounting for around 50% of the sequences obtained. In culture-dependent studies, it seems that fast growing γ-Pro- teobacteria out-grow slower-growing α-Proteobacteria such as rhizobia.

Endophytic bacteria are found in legume nodules as well. In red clover nodules, some species of rhizobia were  found, in- cluding Rhizobium (Agrobacterium) rhizogenes, in addition to R. leguminosarum bv. trifolii, which is the normal clover sym- biont (Sturz et al. 1997). Some  γ-Proteobacteria are cooccu- pants with the specific  rhizobia in Hedysarum plant nodules (Benhizia et al. 2004). In most cases, the endophytic bacteria are unable to form nodules.

 

Effects of endophytic bacteria and benefits to the plant.

The growth stimulation by the microorganisms can be a con- sequence of nitrogen fixation (Hurek et al. 2002; Iniguez et al.

2004; Sevilla et al. 2001) or the production of phytohormones, biocontrol of phytopathogens in the root  zone (through pro- duction of antifungal or antibacterial agents, siderophore pro- duction,  nutrient  competition   and  induction  of  systematic acquired host resistance, or immunity) or by enhancing avail- ability of minerals (Sessitsch et al. 2002a; Sturz et al. 2000). The elucidation  of the mechanisms promoting plant growth

 

 

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will help to favor species and conditions that lead to  greater plant benefits. Volatile substances such as 2-3 butanediol and aceotin produced by bacteria seem to be a  newly discovered mechanism responsible for plant-growth promotion (Ryu et al.

2003). It would be interesting to determine if volatiles could be produced inside plants. Endophytes produce adenine  ribosides that stimulate growth and mitigate browning of  pine tissues (Pirttilä et al. 2004).

Endophytic bacteria of red clover seem to be responsible for the allelopathic effects observed with these plants over maize, causing reduced plant emergence and plant height (Sturz and Christie 1996). For this reason, it was not  recommended to grow maize after clover in Canada. Clover endophytic bacteria reproduced the deleterious effects on  maize. It would have been more convincing if the authors had tested the effects of clover with and without endophytes on maize germination and development.

A recent review on the mechanisms of biocontrol of  plant growth-promoting   rhizobacteria  includes   rhizospheric  and endophytic bacteria (Compant et al. 2005). Bacterial endophytes are  capable of  suppressing  nematode proliferation  and  this may benefit other crops in rotation with the host plants (Sturz and Kimpinski  2004). The frequent isolation of Curtobacte- rium flaccumfaciens as endophytes from asymptomatic citrus plants infected with the pathogen Xylella fastidiosa suggested that the endophytic bacteria may help citrus plants to better re- sist the pathogenic infection (Araujo et al. 2002). Endophytes from potato plants showed  antagonistic activity against fungi (Berg et al. 2005a;  Sessitsch et al. 2004) and also inhibited bacterial pathogens belonging to the genera Erwinia and Xan- thomonas (Sessitsch et al. 2004). Some of the endophytic iso- lates produced antibiotics and siderophores in vitro (Sessitsch et al. 2004).

Inhibition of the oak wilt pathogen Ceratocystis fagacearum was  obtained  with 183 endophytic bacteria  of 889 isolates tested (Brooks et al. 1994). Of 2,648  bacterial isolates ana- lyzed from the rhizosphere, phyllosphere, endosphere, and en- dorhiza, only one, a root endophyte corresponding to Serratia plymuthica, was a highly effective fungal antagonist (Berg et al. 2005a). Endophytic actinobacteria are effective antagonists of the pathogenic fungus Gaeumannomyces graminis in wheat (Coombs et al. 2004), and several endophytes showed antago- nism against Rhizoctonia solani (Parmeela and Johri 2004). It is worth considering that most of the assays to test antagonism are in vitro and it remains to be established if this correlates to effects in nature.

A near-future application may consider the use of geneti cally engineered endophytes with biological control potential in agri- cultural crops. The endophytes Herbaspirillum serope dicae and Clavibacter xylii have been genetically modified to produce and excrete the δ-endotoxin of Bacillus thuringensis to control insect pests (Downing et al. 2000; Turner et al. 1991).

Bacteria degrading recalcitrant compounds are more  abun- dant among endophytic populations than in the rhizosphere of plants  in  contaminated  sites  (Siciliano  et  al.  2001),  which could mean that endophytes have a role in metabolizing these substances.   Engineered   endophytic   Burkholderia   cepacia strains improved phytoremediation and promoted plant tolerance to toluene (Barac et al. 2004). There is an increasing interest on genetically modifying  endophytes (Andreote et al. 2004). The advantages and obstacles to use bioengineered endophytes have been clearly discussed (Newman and Reynolds 2005; van der Lelie et al. 2004).

Endophytic bacteria possess the capacity to solubilize phos- phates, and it was suggested by the authors that the endophytic bacteria from soybean may also participate in phosphate assimi- lation (Kuklinsky-Sobral et al. 2004).

 

Brazilian sugarcane plants have been grown for many years with small amounts of fertilizer without showing symptoms of N deficiencies. Out of the many N2-fixing endophytes isolated from sugarcane, it has not been clearly defined which are re- sponsible for fixing N inside the plant. However, there is con- troversy on the level of N fixed by endophytes and the propor- tion contributed to the plant (Giller and Merckx 2003). These estimates vary  widely in different reports and range from 30 up to 80  kg N/ha/year (Boddey et al. 1995). Under optimal conditions, some plant genotypes seem to obtain part of their N requirements from nitrogen fixation.

Kallar grass grows in N-poor soils in Pakistan and a diver sity of Azoarcus spp. have been recovered from it (Reinhold-Hurek et al. 1993). Inside wheat, Klebsiella sp. strain Kp342 fixes N2 (Iniguez et al. 2004), and it has been reported that it increases maize yield in the field (Riggs et al 2001). Similarly, nitrogen- fixing endophytes seem to relieve N deficiencies of sweet potato (Ipomoea batatas) in N-poor soils (Reiter et al. 2003).

Grasses growing in nutrient-poor sand dunes contain mem- bers of genera Pseudomonas, Stenotrophomonas  as  well as Burkholderia. It seems that the Burkholderia endophytes could contribute N to the grasses, because nitrogenase was detected with antibodies in roots within  plant cell walls of stems and rhizomas (Dalton et al. 2004).

Some research has been directed to find endophytes  that could significantly increase the yields in different crops after their inoculation. To reveal the effects of endophytes, inocula- tion experiments have been performed, but it has been a prob- lem  to  eliminate   resident  or  indigenous  endophytes  from plants in order to have bacteria-free plants or seeds. Functional redundancy  of  resident endophytes and added  inocula may limit the effects observed from inoculation. Very complex mi- crobial community-plant interaction, poor rhizosphere compe- tence with endogenous microorganisms (Sturz et al 2000), and bacterial fluctuations with environmental conditions may also limit the applicability of endophyte  inoculation in the field (Sturz and Nowak 2000).  Furthermore, in the field, the large abundance and diversity of soil bacteria may be a rich source of endophytes and, for this, inoculation effects may not be ob- served. Since surface disinfection does not remove endophytes, procedures such as warming and drying seeds  have been as- sayed to diminish bacterial populations  inside (Holland and Polacco 1994). Tissue culture has also been used to eliminate or reduce endophytes (Holland and Polacco 1994; Leifert et al.

1994). Inoculants seem to be successful in  micropropagated plants, as there are few or no other microorganisms with which to compete. There could be  enormous benefits to be gained through the inoculation of microorganisms into soil-less mixes in  which  plants  are  transplanted at  an  early  stage  in  their growth. In such cases, when the plantlets were inoculated, they were more vigorous and had increased drought resistance, an increased  resistance  to  pathogens,  less  transplanting  shock, and lower mortality (Barka et al. 2000; Martínez et al. 2003; Sahay and Varma 1999).

 

Plant colonization.

Methylotrophs  are  seedborne  in  soybean  (Holland   and Pollaco 1994). Many seeds carry a diversity of  endophytes (Coombs and Franco 2003b; Hallmann et al. 1997). By being seedborne,  endophytes assure their  presence in new plants. Plants that propagate vegetatively (such as potatoes or sugar- cane) can transmit their endophytes to the next generation and would not require the infection process described below. Some pathogens  are  also  found  inside  seeds  (Berg  et  al.  2005b; Schaad et al. 1995).

In the rhizosphere there is a selection of the microorganisms that are able to survive in the root exudates and compete with

 

 

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others.   Rosenblueth   and   Martínez-Romero  (2004)   found strains  that  were  equally  competitive  for   colonizing  the rhizosphere and inside tissues of the root.

In order to colonize the plant, some bacteria must find their way through cracks formed at the emergence of lateral roots or at the zone of elongation and differentiation of the root. Dong and associates (2003) showed that cells of Klebsiella sp/ strain Kp342 aggregate at lateral-root junctions of wheat and alfalfa. Similarly, Gluconacetobacter diazotrophicus and Herbaspiril- lum  seropedicae also colonize lateral-root junctions in high numbers (James and Olivares 1997). Possible infection  and colonization sites have been illustrated by Reinhold-Hurek and Hurek (1998b). It has been proposed that cellulolytic and pec- tinolytic enzymes produced by endophytes are involved in the infection  process  (Hallmann  et  al.  1997),  as  in  Klebsiella strains, pectate lyase has been implicated to participate during plant  colonization (Kovtunovych et al. 1999). The cell wall– degrading   enzymes   endogluconase   and   polygalacturonase seem  to  be  required  for  the  infection  of  Vitis  vinifera  by Burkholderia sp. (Compant et al. 2005).

In some assays, early endophytic colonization differed from one cultivar to another, but later endophytes were recovered in approximately  similar  numbers  from  the  different  cultivars (Pillay  and Nowak 1997). Fungal  colonization could affect colonization by endophytic bacteria (Araujo et al. 2001) or the reverse could be true.  Strain to strain variation in colonizing capabilities  have  been  found  among  Rhizobium  etli  strains (Rosenblueth and Martínez-Romero 2004), and these may be related to  differences in genomic content (Rosenblueth et al. unpublished data). In general, endophytic isolates were capa- ble of colonizing or recolonizing the inside plant  tissues in higher numbers than isolates from the root surface (van Peer et al. 1990; Rosenblueth and Martínez-Romero 2004).

After 1 day of exposure of certain plant roots to  bacteria, they can be found in the aerial parts of the plant. Guo and as- sociates (2002) inoculated the roots of hydroponically grown tomato plants with salmonellae at  around 4.55 log CFU ml–1

and,  the  next  day,  found  that  hypocotyls,  cotyledons,  and

stems had around 3 log CFU g–1. The systematic spread of an endophytic Burkholderia strain to aerial parts of Vitis vinifera seems to be through the transpiration stream (Compant et al.

2005).

Solomon and Matthews (2005) irrigated lettuce plants with Escherichia  coli  0157:H7 or  with  fluorescent  microspheres (used as bacterial surrogates). After 1, 3, and 5 days, there was no significant difference (P ≤ 0.05) in  populations between days, but there was significant difference in the internalization of bacteria or fluorescent  microspheres. It is remarkable that microspheres attain  100-fold higher levels in plants than do Escherichia  coli  cells  (Solomon  and  Matthews  2005). The authors suggested that the entry of Escherichia coli into plant roots is not determined by specific bacterial factors but, rather, by the plant.

Endophytes can also play an  active role in  colonization. Azoarcus sp. type IV pili are involved in the adherence to plant surfaces,  an  essential  step  towards  endophytic  colonization (Dörr et al. 1998). Two Klebsiella strains differ significantly in their invasion capacity in different plant hosts (Medicago sa- tiva, Medicago truncatula, Arabidopsis thaliana, Triticum aes- tivum, and  Oryza sativa). One of them (Kp342) was a better colonizer in all hosts and only needed a single cell to colonize the plants substantially a few days after inoculation (Dong et al. 2003). The plant hosts also  differed in their ability to be colonized endophytically by the same bacterium, further sug- gesting an active host role in the colonization process.

Some rhizospheric bacteria can colonize the internal  roots and stems, showing that these bacteria are a source for endo-

 

phytes (Germaine et al. 2004), but also phyllosphere bacteria may be a source of endophytes (Hallmann et al. 1997).

Some flavonoids increased by almost 100% the number of lateral root cracks colonized in Arabidopsis thaliana by Her- baspirillum    seropedicae   and    Azorhizobium    caulinodans (Webster et al. 1998). Colonization of wheat by Azorhizobium caulinodans  and Azospirillum brasilense  was stimulated by flavonoids (Webster et al. 1998), as  was the colonization by Azorhizobium  caulinodans  of  two  Brassica  napus  (oilseed rape) varieties (O’Callaghan  et al. 2000). In Rhizobium spp., plant flavonoids were involved in inducing mechanisms to re- sist plant-defense phytoalexins (González-Pasayo and Martínez- Romero  2000;  Parniske et  al.  1991).  Flavonoids  are  better known for their role in inducing the expression of nod genes that code for enzymes producing Nod factors.  Neither Nod genes nor Nod factors are required for the endophytic coloni- zation of Arabidopsis thaliana or  wheat (Gough et al. 1997; Webster et al. 1998). Therefore, the role of flavonoids in stimu- lating colonization may be related to regulating other bacterial genes, such as those for phytoalexin resistance, type III secre- tion (Perret et al.  1999; Viprey et al. 1998), or genes for the synthesis of  lipopolysaccharides (Reuhs et al. 2005), partici- pating in the interaction with the plant. Interestingly, a glucosi- dase  enzyme hydrolyzing glucoside isoflavones was purified from an endophytic Pseudomonas sp. (Yang et al. 2004). This activity could contribute to produce active (aglycone) flavons inside plants, but this has not been tested.

Changes in plant physiology can lead to the development of a distinct endophytic population (Hallmann et al. 1997). A di- minished colonization of sugarcane by Gluconacetobacter di- azotrophicus was observed in plants under a high nitrogen-fer- tilization regime as opposed to  low N fertilization. It seems that supplying nitrogen to the plants alters its physiology and may cause a decrease in sucrose, which seems to be used for the endophytic growth (Fuentes-Ramírez et al. 1999). Fertilizer effects over endophytic populations were further confirmed. In rice, a rapid change of the nitrogen-fixing population was ob- served within 15 days after nitrogen fertilization (Tan et  al.

2003). Organic amendments to plants also influence the endo- phytic populations (Hallmann et al. 1997).

Colonization does not depend on the nitrogen-fixing ability of the bacteria, as Nif –   mutants of  Gluconacetobacter diazo- trophicus or Herbaspirillum  seropedicae, were able to colo- nize  as  well  as  Nif +   strains  (Roncato-Maccari et  al  2003; Sevilla et al. 2001). In contrast, Azoarcus mutants affected in pili were incapable of systemic spread into rice shoots (Dörr et al. 1998); this is also the case with mutants unable to produce

a secreted endoglucanase (Reinhold-Hurek et al. 2006). Non- motile mutants of Salmonella enterica were incapable of colo- nizing or had only a reduced invasion capacity in Arabidopsis thaliana (Cooley et al. 2003).

As plants have a determinant role in controlling endophytic colonization, it is important to avoid performing colonization assays in the laboratory with plants under suboptimal growth conditions,  as they may  show unbalanced interactions with endophytes with occasional overestimation of bacterial coloni- zation  by  some  strains. An  increased diversity  of  bacterial endophytes was found in Erwinia carotovora–infected  pota- toes in comparison with noninfected control plants (Reiter et al. 2002). The colonizing capacity may also be overestimated in vitro, as there is no competition with indigenous soil bacteria (Cooley et al. 2003).

 

Plant location.

The methods used to assess the occurrence and location of endophytic bacteria have been diverse and include  immuno- logical detection of bacteria, fluorescence tags,  and confocal

 

 

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laser   scanning   microscopy  (Chelius  and  Triplett   2000b; Hartmann et al. 2000; Verma et al. 2004). In addition, specific oligonucleotide probes could be of use  to analyze bacteria inside plants (Hartmann et al. 2000).

Endophytic bacteria are found in roots, stems, leaves, seeds, fruits, tubers, ovules, and also inside legume nodules (Benhizia et al. 2004; Hallmann et al. 1997; Sturz et al. 1997). In most plants, roots have the higher numbers of endophytes compared with above-ground tissues (Rosenblueth and Martínez-Romero

2004).

It seems that the bacterial endophytes described here do not inhabit living vegetal cells (James and Olivares 1997; Reinhold- Hurek and Hurek 1998a). Intercellular spaces and xylem vessels are the most commonly reported locations for endophytic bac- teria  (Reinhold-Hurek and Hurek 1998a;  Sprent and James

1995). A Burkholderia sp. strain was found in xylem  vessels and substomatal chambers in Vitis vinifera plants (Compant et al. 2005). By inhabiting similar niches as vascular pathogens, endophytes  may be used as  competing bacteria for disease control (Hallmann et al. 1997).

Surface-washing  of  sprouts  was  not  an  effective  way  to eliminate  Salmonella  enterica  and  Escherichia  coli  strains from alfalfa sprouts or seeds, indicating that these bacteria are located in a protected niche (Cooley et al. 2003). By introduc- ing the green fluorescent protein,  their location was defined; Salmonella  enterica  colonized  seed  coats  and  roots,  while Escherichia coli colonized only roots (Charkowski et al. 2002).

 

Molecular interactions.

In contrast to the extensive information on the  molecular mechanisms of other bacteria-plant interactions (Lugtenberg et al. 2002; Oldroyd and Downie 2004), there is only limited data on the endophyte-host molecular interactions.

The plant response. The plant response to endophytes seems to be conditioned, to a large extent, by the plant  genotype. Wild races and some plant varieties seem to provide adequate in-plant conditions that stimulate and support nitrogen fixation by endophytes and benefit from  it. This has been observed with rice, sugarcane, and maize (Boddey et al. 1995; Engelhard et  al.  2000;  Gutiérrez-Zamora  and  Martínez-Romero 2001; Iniguez et al. 2004; Reis et al. 1994; Shrestha and Ladha 1996; Urquiaga et al. 1989).

Plant genes may be modulated by the presence of the bacte- ria, and the genes so expressed provide clues as to the effects of endophytes in plants. In sugarcane, genes expressed in re- sponse to the endophytic colonization of  Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans are being studied (de Matos Nogueira et al. 2001). The sequence analy- sis of the cDNA and other  libraries derived from messenger RNAs expressed in sugarcane when inoculated with Glucona- cetobacter  diazotrophicus  and  Herbaspirillum  rubrisubalbi- cans revealed that genes for nitrogen assimilation, for carbon metabolism, for plant growth, as well as genes for a  limited plant defense were induced (de Matos Nogueira et al. 2001). Similar strategies could be followed to study  the effects of other endophytes in plants.

Arabidopsis thaliana  has been used as a  model plant  to study its interactions with endophytes like Azorhizobium cauli- nodans and enterobacteria. The advantage of using Arabidop- sis thaliana is that there are defined mutants that may be tested for their colonization by endophytes.  Iniguez and associates (2005)  used Medicago truncatula  and Arabidopsis thaliana mutants to determine the role of plant defense-response path- ways in regulating the  number of endophytic bacteria. They found that ethylene, a signal molecule for induced systemic re- sistance in plants, decreases endophytic colonization by Kleb- siella sp. strain Kp342 and Salmonella enterica serovar Typhi-

 

murium (Salmonella typhimurium) strains. An ethylene-insen- sitive  Medicago  truncatula  mutant  was  hypercolonized  by Kp342, while the addition of the ethylene precursor 1-amino- cyclopropane-1-carboxylic acid to wild-type Medicago trunca- tula and wheat reduced the amount of endophytic bacteria. By treating the wild-type plants with the ethylene inhibitor 1-me- thylcyclopropane, a reversion of the reduction of colonization was obtained. Iniguez and associates (2005) also found that the presence of bacterial extracellular components, such as flagella and  type  III  secretion  systems  (TTSS-SPI1) of  Salmonella pathogenicity island  1 decrease endophytic colonization, and Salmonella  typhimurium  mutants  lacking  these  components have  higher endophytic colonization in Medicago sativa and wheat  seedlings. Arabidopsis  thaliana mutants showed  that only a salicylic acid (SA)-independent defense response con- tributes  to  restriction  of  the  colonization  by  Klebsiella  sp. strain Kp342. In the case of colonization by Salmonella typhi- murium, this is restricted by both SA-dependent and –independ- ent pathways. The study of  Salmonella typhimurium flagella mutants suggests that flagella are recognized through the SA- independent  response and TTSS-SPI1 is  recognized by the SA-dependent response that is involved in inducing the  pro- moter  of  PR1,  a  SA-dependent  pathogenesis  related  gene. Kp342 lacks flagella and TTSS-SPI1 (Dong et al. 2001); thus, when it associates with the plant, it  does not induce the SA- dependent  responses  and  may  colonize the  plant in  higher numbers (Iniguez et al. 2005). Kp342 might have lost its fla- gella during its evolution in association with plants (Iniguez et al. 2005). Other bacteria that interact closely with plants, such as Rhizobium and Agrobacterium spp., have flagella, but these are not  elicitors  of  plant  defense mechanisms (Felix et  al.

1999). In plants as well as in mammals and insects, bacterial flagellins are recognized by surface receptors that contain trans- membrane proteins with extracellular  leucine-rich repeat do- mains (Gómez-Gómez and Boller  2002). Flagellin acts as an elicitor in whole Arabidopsis thaliana plants, inducing an oxi- dative burst, callose  deposition, and ethylene production and leading to the  induction of defense-related genes. Plants can detect the presence of molecules from bacteria through chemo- perception systems (Boller 1995).

A local host-defense reaction was induced by an endophytic

Burkholderia strain in Vitis vinifera plants (Compant et  al.

2005). It remains to be determined if endophytes are affected by innate immunity in plants, as occurs in some animal-bacte- rial interactions (McPhee et al. 2005). Antimicrobial peptides have been isolated from maize and  rice (Duvick et al. 1992) and could have a role in  controlling endophytic populations. Limiting carbon  sources in the carbon apoplastic fluid may also restrict endophytic growth (Fuentes-Ramírez et al. 1999).

Bacterial genes expressed in the presence of plants.  Endo- phytes may differentially express genes that are required to en- ter and colonize the plant to grow and  survive within plant tissues and to stimulate plant  growth,  compete and suppress pathogens, or produce different substances.

Endophytic bacteria provide useful and rich models to study the genetic expression of bacteria in their natural niches or habi- tats (inside plants), which are more structured and variable than culture media under controlled laboratory conditions. Neverthe- less, very little work has been done on this. Genomic projects are being performed on some endophytic bacteria, such as Azo- arcus spp. (Battistoni et al. 2005), Herbaspirillum sp., Glucona- cetobacter diazotrophicus, and Klebsiella spp., which will cer- tainly be of great help to further understand their  molecular interaction with plants. In vivo expression technol ogy used to study gene expression in different niches, (Rediers et al. 2005) including the rhizosphere (Ramos-González et al. 2005), may be used as well to study gene expression during endophytic life.

 

 

832 / Molecular Plant-Microbe Interactions

 

Roncato-Maccari  and  associates  (2003)  examined  micro- scopically  a  Herbaspirillum seropedicae  Nif +   strain  with a gusA cassette in its nifH gene after inoculation of maize, sor- ghum, wheat, and rice. They found expression of nif genes in bacterial colonies located in external mucilaginous root mate- rial eight days after inoculation, as well as in roots, stems, and leaves. Using a  similar approach, Vermeiren and associates (1998) had previously found that nifH gusA fusions of Pseudo- monas  stutzeri (formerly Alcaligenes faecalis) and Azospiril- lum  irakense were also expressed in rice roots. It was also observed that the NifH (nitrogenase reductase) of  Klebsiella pneumoniae occurred in maize roots but not in stems, using an antibody to the purified enzyme (Chelius and Triplett 2000b). In situ hybridization studies demonstrated that Azoarcus nitro- genase  genes are  expressed inside the roots of field-grown Kallar grass  (Hurek et al 1997). These results suggest that grass tissues provide a suitable environment for nitrogen-fixa- tion gene expression, but it seems that N2   fixation in the plant is carbon-limited (Christiansen-Weniger et al 1992). Different grasses inoculated with members of genera  Herbaspirillum, AzospirillumKlebsiella, and  Serratia  produced ethylene in acetylene reduction assays only  when a carbon source was added  (Chelius  and  Triplett   2000b;  Egener  et  al.  1999; Gyaneshwar et al 2002).

Bacteria  can  communicate  through  “quorum  sensing,”  a term applied to the production of diffusible signal  molecules that control gene expression in a manner dependent upon bac- terial population density (quorum)  (Swift et al. 1996). Until now, phytopathogenic bacteria have been reported to respond to quorum to produce antibiotics, virulence factors, and plant cell wall–degrading  exo-enzymes (Von Bodman et al. 2003). Interestingly,  plants  can  perceive “quorum  sensing”  signals from the  bacteria and control quorum-regulated bacterial re- sponses (Bauer and Mathesius 2004; Mathesius et al. 2003). It would be interesting to determine if endophytes produce “quo- rum  sensing”  molecules  inside  plants  and  the  effects  they cause.  Inside  the  plant,  there  could  be  exchange  of  signal molecules among  microorganisms  and  of  bacteria  with the host, although this has not yet been reported.

It would also be interesting to address whether some of the well-studied molecular mechanisms used by  phytopathogenic bacteria (Van’t Slot and Knogge 2002)  are to some extent shared with endophytes.

 

May endophytes be or become pathogens?

Most  fungal  grass  endophytes are  considered  mutualistic with their hosts. The main advantage for the plants is the pro- tection  they  confer  against  herbivory   by  toxic  alkaloids (Schardl et al. 2004). These fungal endophytes, like bacteria, may  obtain  nutrients  from  the  plant  and  protection  from abiotic stresses like desiccation. But some fungal endophytes (Stone et al. 2000) may become plant pathogens, depending on the  developmental stage of host and fungus, environmental factors, and host defense responses (Schulz and Boyle 2005). It was frequently found that some bacterial endophytic isolates from healthy plants inhibited the growth of tomato seedlings in reinoculation assays, possibly  through the production of cer- tain metabolites (van Peer et al. 1990). Some endophytes seem to be latent pathogens, and infections may proceed under cer- tain conditions. These may be due to changes in environmental conditions  such as CO2    accumulation or O2    depletion (Lund

and Wyatt 1972), but others could be related to the presence of

other microorganisms interacting with the endophyte.  There are reports that mixed inoculations of two endophytic bacteria that individually inhibit growth result in plant-growth promo- tion (Sturz et al. 1997). The order in which endophytic popula- tions are inoculated and become established in the host plant

 

could affect subsequent plant-growth promotion effects (Sturz and  Christie  1995).  Herbaspirillum   rubrisubalbicans  may cause a mild mottled stripe disease in a few sugarcane varieties but give no symptoms in most hosts (James et al. 1997; Olivares et al. 1997). It seems there is an equilibrium of endophytes and plants that under certain circumstances may be unbalanced to the detriment of one of the partners.

In other cases, endophytes have been found to be closely re- lated to human pathogens or are either human or opportunistic human pathogens. This is the case of  endophytic Salmonella strains, which have caused  outbreaks and constitute a health risk for consumers of  raw fruits and vegetables (Guo et al.

2002), and of the Burkholderia cepacia strains isolated  from plants (Barac et al. 2004). As Burkholderia  cepacia causes pulmonary infection (even fatal) in human  cystic fibrosis pa- tients, a reassessment of the risk and a moratorium on the agri- cultural  use  of  Burkholderia   strains  have  been  suggested (Holmes et al. 1998; Parke  and Gurian-Sherman 2001). Ap- proaches to reduce raw vegetable contamination produced by pathogens propose  strategies to increase the number of safe growth-promoting bacteria in plants (Iniguez et al. 2005).

Nocardia endophytes have been isolated from members  of genus Citrus. Some Nocardia species are known to be human pathogens causing nocardiasis (a severe human  infection in feet and legs that may lead to amputation) that is transmitted by soil. A fluctuating and prominent population of Mycobacte- rium spp. was found in Scots pine that seemed to be required for bud development. When the tissue was fully developed, the endophyte was no longer detected (Pirttilä et al. 2005). Myco- bacterium isolates related to those reported as clinical isolates were  the most frequently encountered in wheat in Australia (Conn and Franco 2004). It was not analyzed whether  these mycobacteria carry some virulence genes.

Parke and Gurian-Sherman (2001) stated “It is not  coinci- dental  perhaps  that  many  of  the  most  effective  biocontrol agents (Stenotrophomonas maltophilia, Pantoea agglomerans,

… and Burkholderia cepacia) of plant diseases are also oppor- tunistic human pathogens. These … are fiercely  competitive for nutrients and may produce antimicrobial  metabolites and may themselves be resistant to multiple  antibiotics.” In addi- tion, surviving plant defense reactions may render endophytic bacteria resistant to human  defense responses as well. Some endophytes have been found to contain genes that are required for  virulence in  pathogens  (Barak  et  al.  2005;  Dörr  et  al.

1998). It has been difficult to differentiate environmental iso- lates and clinical bacteria of Pseudomonas spp.  (Foght  et al.

1996). This is the case with plant endophytes from rice, sugar- cane, banana, and maize (Martínez et al. 2003)  and  clinical isolates from hospitals in Mexico  (Rosenblueth et al. 2004) and in Europe (Brisse and Verhoef 2001). All these were clas- sified  as  Klebsiella   variicola  (Rosenblueth  et  al.  2004). Although  Klebsiella  variicola seems to be less virulent than Klebsiella pneumoniae and has different epidemiological  dy- namics (Martínez et al. 2004), its use in agriculture  was dis- couraged (Lloret et al. 2004). Even if some plant bacterial line- ages  may  be  distinguished  from  pathogenic  lineages,  their close genetic relatedness may  render the former good recipi- ents to acquire virulence  genes by lateral transfer from their pathogenic relatives.  Still very little is known of the genetic behavior of bacteria and the frequency of transfer of genes in natural habitats.

 

Perspectives.

The natural condition of plants seems to be in a close inter- action with endophytes. Endophytes seem promising to increase crop yields, remove contaminants, inhibit pathogens, and pro- duce fixed nitrogen or novel substances. The repertoire of their

 

 

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effects and functions in plant has not been  comprehensively defined. The challenge and goal is to be able to manage micro- bial communities to favor plant colonization by beneficial bac- teria. This would be  amenable when a better knowledge on endophyte ecology and their molecular interactions is attained. The  contributions of this research field may have economic and environmental impacts.

 

ACKNOWLEDGMENTS

 

Thanks to J. Martínez for technical support and to M.  Dunn and P. Vinuesa for reading the manuscript. Partial  financial support was from CONACyT grant 40997-Q.

 

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