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The phyllosphere is a complex habitat hosting diverse microorganisms and its

study requires thorough methodology. The T-RFLP technique combined with HhaI is

a powerful tool to study the barley phyllosphere. Unlike the RISA method, it allows

an estimation of the community structures (ecological indices), group individuals

(PCA) and focus on particular OTUs of interest. However, the molecular profiling

can be affected by many experimental factors. Here, the microbial removal method

was shown to significantly modify the structure of the epiphytic populations. All

69 compared to the buffer alone (Figure 3.2), suggesting that they were able to disrupt

the bacterial biofilms and/or other microbial adhesion mechanisms differentially.

A biofilm is a complex structure, mostly formed of polysaccharides held tight

by cations such as calcium (Ca2+) (Sutherland, 2001; Morris and Monier, 2003). It

provides many advantages such as an anchor to the leaf surface and protection

against UV and osmotic shifts (Davey and O'Toole, 2000). Hence, bacteria trapped

in biofilms may not be effectively removed from the leaf surface. Both EDTA and

hypochlorite treatments promoted the removal of bacteria, but also filamentous fungi

from the leaf surface. They were shown to cause partial removal of biofilm proteins

in a bioreactor (Chen and Stewart, 2000): EDTA is supposed to disrupt the biofilm

structure by chelating cations, such as Ca2+, essential to biofilm integrity (Turakhia et

al., 1983), whereas hypochlorite causes biofilm protein removal by an unknown

mechanism (Chen and Stewart, 2000).

Another way to promote microbial removal consists of gently disrupting the

leaf surface. Surfactants improved the isolation of both bacteria and filamentous

fungi, but Triton and SDS hindered yeast isolation. Anionic surfactants (SDS) were

shown to solubilise the leaf epicuticular waxes (Tamura et al., 2001), whereas

nonionic surfactants (Tween and Triton) induced a reduction of the stiffness of plant

wax surface (Grant et al., 2008). However, using chemically similar surfactants

(Tween and Triton) did not result in similar effects on the whole populations. The

surfactant concentration also affected differentially the extraction. Nevertheless, the

overall proportions of the bacterial populations were conserved, no matter what

surfactant was used (Figure 3.6). Even though adding chemicals to the isolation

buffers improved the global isolation of microorganisms, certain of them were

70 abundance of a few OTUs, suggesting either an improved or impaired removal

(Figure 3.6). Furthermore, the diversity of the epiphytic population significantly was

reduced (Table 3.5), which means that the population is more dominated by a few

microbes. As the aim of this study was to investigate the complex ecology of the

phyllosphere, no extra chemicals were added to the buffer. The technique used also

affected differentially the outcome of the isolation. Sonication was better than

washing: as leaves had to be roughly chopped to fit in the flask for washing,

epiphytic solutions were green, indicating the presence of chlorophyll and other plant

debris. To avoid plant DNA to be detected on the T-RFLP profiles, the sonication

was preferred to the washing method and no surfactant or other chemical was added

to the extraction buffer.

The barley phyllosphere of Sumo harboured many microorganisms: mostly

bacteria, then yeast and filamentous fungi, reaching over one million individuals per

gram of fresh leaf weight (Figure 3.2). Bacteria are usually described as the most

common organism on the leaf surface (Lindow and Brandl, 2003), and the fungal

population, dominated by ascomycota, was much larger on older leaves (Thompson

et al., 1993; Jumpponen and Jones, 2009; Glushakova and Chernov, 2010), which

was consistent with our findings (Figure 3.2 and 3.9). Overall, more bacteria were

observed on barley leaves by Fountaine et al. (2009), but these leaves were much

younger than the ones used in this study. This difference in the number of bacteria

between young and old leaves was in contradiction with the findings of Thompson et

al. (1993) on sugar beet, where it was shown that older leaves have more bacteria

than young ones. However, these two analyses cannot be directly compared with

each other as they are based on different cultivars and different locations. Significant

71 various rice cultivars (de Costa et al., 2006a). Even Fountaine et al. (2009) found

significant differences between the two monitored cultivars: Optic and Cellar.

The Pseudomonas bacteria were the most common culturable bacteria, which

was consistent with the literature (Rotem et al., 1976; Ercolani, 1991; Thompson et

al., 1993). They have many different roles: P. syringae strains are well-known

pathogens and ice nucleation-active bacteria (Hirano and Upper, 2000), whereas

some P. fluorescens strains can be pathogenic (Cui et al., 2005) or act as a BCA

(Paulsen et al., 2005). The other two Pseudomonas species have no known role: P.

graminis was identified on grasses (Behrendt et al., 1999) and P. migulae in water

streams (Verhille et al., 1999). Erwinia tasmaniensis is a potent BCA on apple trees

against the fire blight pathogen, caused by the closely related bacterium Erwinia

amylovora (Geider et al., 2006). The roles of these culturable bacteria on barley

leaves are unknown, but could potentially interact with disease causal agents.

The diffusates from a fluorescent Pseudomonad strain isolated from R.

commune lesions were shown to induce total lysis of R. commune protoplasts, but

lysis of spores was achieved from diffusates from R. commune-infected leaf lesions,

suggesting complex interactions between microorganisms (Rotem et al., 1976).

Furthermore, culturable bacteria represent only a fraction of the whole leaf-

associated population (Yang et al., 2001). The ratio between culturable and

unculturable bacteria (estimated using DGGE) from the rhizosphere has been shown

to vary between 0.1 and 10% depending on the soil type (Torsvik et al., 1998).

Similarly, T-RFLP profiles revealed that the identified culturable bacteria represent

only a fraction (up to 24%, Annex Table A.6) of the total community, which was of a

similar order to what Wilson and Lindow (1992) observed with P. syringae on bean

72 The role and, most importantly, the identity of other unculturable bacteria of

the Sumo leaves are still unknown. The OTUs corresponding to the culturable

bacteria were easily found in silico and validated experimentally. The identification

of other OTUs from databases, such as MiCA (Shyu et al., 2007), is not possible.

The most common OTU (62; Annex Table A.6) matched only seven different

bacterial species, none of which were known plant-associated bacteri (data not

shown). Furthermore, predicted and experimental TRF size did not match perfectly

(Table 3.8). Using the MiCA database, the 61, 62 and 63 OTUs corresponded to over

80 different genera and more than 1,300 accession numbers. A clone library would

be essential to identify the missing OTUs, but no interaction study could be

undertaken without culturable isolates.

The roles of fungi also should not be ignored. As the plant ages they represent

an increasing proportion of the whole microbial community (Thompson et al., 1993;

Jumpponen and Jones, 2009; Glushakova and Chernov, 2010). Many yeast, when

inoculated on barley leaves before challenge with the causal agent of the barley leaf

scald, induced a significant increase of visible symptoms (Fountaine et al., 2009).

Such knowledge could help to develop more integrated crop protection strategies, to

not only kill causal agents, but also helper microorganisms (Newton and Toth, 1999;

73

CHAPTER 4: INTERACTIONS BETWEEN MICROORGANISMS