Wei Liu

Xiao-Gang Lu

Pascal Boulet

Marie-Christine Record

Influencing factors of atomic order in the binary sigma phase

Keywords: sigma phase, atomic order, atomic charge, size factor, number of valence electrons, total number of electron shells

The present work brings new insights into the influencing factors of atomic order (i.e. atomic constituent distribution or site occupancy preference on inequivalent sites of a crystal structure) of the binary sigma phase. We have calculated the volumes and atomic charges of the constituent elements of 32 stoichiometric end-member compounds (i.e. the complete set of ordered configurations) of the binary sigma phase by using first-principles calculations. The calculation results show that besides size factor and the number of valence electrons, the total number of 2 electron shells is found as the third factor that affects the atomic order of the sigma phase.

Moreover, the calculation results indicate that between the two constituent elements, the one with a larger atomic size, smaller number of valence electrons or smaller total number of electron shells prefers occupying large coordination number (CN) sites (i.e. 4f, 8i 1 , 8j) and the other prefers occupying small CN sites (i.e. 2a, 8i 2 ). Furthermore, we have dissociated the effect of individual influencing factor on atomic order of the sigma phase combining with the measured site occupancies from the literature.

INTRODUCTION

The sigma phase is a non-stoichiometric intermetallic compound that crystallizes in a tetragonal structure (space group P4 2 /mnm) with 30 atoms distributed on five inequivalent sites, namely 2a, 4f, 8i 1 , 8i 2 and 8j 1-3 . It is a hard, brittle phase mostly formed between transition elements. It deteriorates various properties of stainless and other high-alloy steels as well as Ni-based superalloys 1-2 . The investigation of the sigma phase has attracted many interests since 1950s 4 . There are numerous investigations on the atomic order (i.e. atomic constituent distribution or site occupancy preference on inequivalent sites of a crystal structure) of the sigma phase, e.g. (Ref. 1-2, 5-17), by using XRD (X-ray diffraction), ND (neutron diffraction), mathematical methods or first-principles calculations. At first, Kasper and Waterstrat 6 indicated that 2a and 8i 2 sites should be occupied by elements to the right of Mn in the periodic table; 4f site by elements to the left and 8i 1 and 8j sites by a mixture of both. Spooner and Wilson 8 stated that the size of the constituent atoms was a major factor in governing the filling of 2a, 4f, and 8i 2 sites but in addition some valence electron factor governed the filling of 8i 1 and 8j sites. Afterwards, a systematic review of the sigma phase was conducted by Hall and Algie 1 and then by Joubert 2 . They indicated that atoms with large size or poor in d-electrons preferentially occupy sites with large coordination number (CN), namely 4f, 8i 1 and 8j; atoms with small size or rich in d-electrons preferentially occupy sites with small CN, namely 2a and 8i 2 . However, all the conclusions above cannot explain the measured site occupancies of the sigma phase perfectly. The main reason is that the size factor and the number of valence electrons are not enough to explain the atomic order. Taking Re-Mn system as an example, the numbers of valence electrons of Re (5d 5 6s 2 ) and Mn (3d 5 4s 2 ) are the same so the size factor should be the primary influencing factor on the atomic order. However, large differences can be observed in the site occupancies measured for sites with the same CN (8i 1 vs 8j, CN=14) and thus with similar size, as discussed in the following Section 3.2. In fact, the experimental site occupancies show that the atomic order among the large CN sites (i.e. 4f, 8i 1 , 8j) or between the small CN sites (i.e. 2a, 8i 2 ) can be very large, more examples being Nb-Re (4f vs 8i 1 / 8j) 2 , Re-Fe (8i 1 vs 8j) 2 . The early conclusions 1-2 about the atomic order cannot distinguish among the large CN sites or the small CN sites.

In addition, Sluiter et al. calculated the site occupancies of the sigma compounds in Cr-Fe [START_REF] Sluiter | Site occupation reversal in the Fe-Cr σ phase[END_REF] , Os-Cr [START_REF] Sluiter | Site occupation in the Cr-Ru and Cr-Os σ phases[END_REF] , Re-Ta [START_REF] Berne | Ordering effects in the Re-W and Re-Ta sigma phases[END_REF] , Re-W [START_REF] Berne | Ordering effects in the Re-W and Re-Ta sigma phases[END_REF][START_REF] Berne | Site occupancy in the Re-W sigma phase[END_REF] and Ru-Cr [START_REF] Sluiter | Site occupation in the Cr-Ru and Cr-Os σ phases[END_REF] systems by using a first-principle statistical thermodynamic approach. Their calculation results do not fit the experimental data very well, especially for Cr-Fe [START_REF] Sluiter | Site occupation reversal in the Fe-Cr σ phase[END_REF] and Os-Cr 17 systems because of their simplified assumptions. They confirmed the influencing factors on atomic order of Ru-Cr and Os-Cr systems from the perspective of the crystal structure 4 electronic degeneracy arguments and 3) the pairwise interaction (A-A, A-B or B-B) within the sigma unit cell. It was indicated that their conclusion has no predictive value [START_REF] Sluiter | Site occupation in the Cr-Ru and Cr-Os σ phases[END_REF] . Actually, their argument about the pairwise interaction was based on the deduction that all five crystal site were equivalent as Cr and Ru/Os do not have an overwhelming preference for any of the sites. However, this deduction is not totally correct, which will be discussed in Section 3.2.3.

Besides, Crivello et al. 5 systematically discussed the site occupancy behavior of the sigma phase in binary Re-X (X= Co, Cr, Fe, Hf, Mn, Mo, Nb, Ni, Os, Ru, Ta, Tc, Ti, V, W and Zr) systems using the compound energy formalism (CEF) combined with fist-principles calculations. These studied systems include stable sigma compounds (Re-V, Re-Cr, Re-Mn, Re-Fe, Nb-Re, Mo-Re, Ta-Re and W-Re) and as well as some hypothetically unstable ones (Ti-Re, Zr-Re, Hf-Re, Re-Co, Re-Ni, Re-Tc, Re-Ru and Re-Os). The site occupancies of the above binary sigma compounds were calculated at 2000K (see Fig. 9 in Ref. (5)) and Crivello et al. 5 proposed that the site occupancy behavior of the binary sigma phase is related to whether Re bears a larger size than the other constituent element or not. The site occupancy behavior between the same CN (i.e. CN12: 2a vs 8i 2 ; CN14: 8i 1 vs 8j) sites were not clearly clarified. If Re bears a larger size (e.g. for Re-V, Re-Cr, Re-Mn, Re-Fe, Re-Co, Re-Ni, Re-Tc, Re-Ru and Re-Os systems), the site occupancy preference for Re atom follows 4f>8i 1 >8j>8i 2 >2a and if Re bears a smaller size (e.g. for Nb-Re, Mo-Re, Ta-Re, W-Re, Ti-Re, Zr-Re and Hf-Re systems), the site occupancy preference for Re atom follows 8i 2 /2a>8i 1 /8j>4f. However, for Re-Os, Re-Ru and Re-V systems where Re bears a larger size, the calculation results showed that the site occupancy preference for Re atom follows 8j>8i 1 (for Re-Os and Re-Ru systems) and 8i2>8j (for Re-V system) which is inconsistent with the above conclusion. Besides, form the experimental perspective, for Re-V system the site occupancy sequence follows 8i 2 >2a>8i1>4f>8j (see Section 3) which is almost the opposite sequence as the one proposed by Crivello et al. 5 At present, plentiful site occupancy measurements have been conducted on the binary sigma compounds with high accuracy, but the influencing factors on the atomic order of the sigma compounds are still not clear. Apparently, size factor and the number of valence electrons are not enough for the explanation of the atomic order. Another obstacle to sort out the connections is that the experimental site occupancies are results of competition and cooperation of the influencing factors on atomic order and it is hard to dissociate the effect of individual influencing factor, as sometimes for a specific system, different factors have similar effects 14 (e.g. for Cr-Fe system, larger size and also poorer in d-electrons of Cr comparing to Fe both cause Cr preferentially occupy sites with large CN).

In the present work, besides the size factor and the number of valence electrons, we also considered the total number of electron shells as an influencing factor affecting the atomic order of the sigma compounds. Finally, we dissociated the effect of individual influencing factor on atomic order. It is noteworthy that the present findings have predictive ability. To facilitate explanation, all the binary sigma compounds investigated were designated as A-B where the size of atom A is always larger than that of atom B. The sizes of the constituent elements (A and B) are determined by the molar volumes of their pure elements in the sigma phase structure from the CALPHAD assessments [START_REF] Liu | Modeling of molar volume of the sigma phase involving transition elements[END_REF] . Besides, the site occupancy of atom A was given in the present work.

Atom B complements atom A on different sites. We use Y S (A) to denote the site occupancy of atom A on site S. The site occupancy of atom B on site S is equal to 1-Y S (A). The sequence of atomic order of atom A was given in the present work. For example, the sequence of atomic order, 4f>8i1>8j>8i2>2a, denotes a descending site occupancy preference of atom A from the site 4f to 2a. The sequence of atomic order of atom B is the opposite.

METHODOLOGY AND CALCULATION DETAILS

First-principles calculations

The first-principles calculations were performed using the plane wave method with projector augmented wave (PAW) pseudo-potentials [START_REF] Kresse | From ultrasoft pseudopotentials to the projector augmented-wave method[END_REF] , as implemented in the Vienna ab initio simulation package (VASP) [START_REF] Kresse | Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set[END_REF] . We used the exchange-correlation functional within the generalized gradient approximation (GGA) as parameterized by Perdew and Wang (i.e. GGA-PW91) [START_REF] Perdew | Accurate and simple analytic representation of the electron-gas correlation energy[END_REF] . The magnetic parameter for ferromagnetism was set and spin-polarized calculations were conducted. The structure was fully relaxed and after the relaxation, a final static calculation was performed by adopting tetrahedron method with Blöchl corrections. A large plane-wave cutoff energy of 400 eV was used. The k-point meshes (8*8*15) for Brillouin zone sampling were constructed using the Monkhorst-Pack scheme [START_REF] Monkhorst | Special points for Brillouin-zone integrations[END_REF] . The convergence criteria for electronic self-consistency and ionic relaxation were 10-5 eV/atom and 10-4 eV/atom, respectively. These settings ensure the energy converge to at least 1 meV/atom. To facilitate the calculations, ZenGen script-tool was used to automatically generate input files for VASP calculations 23 .

Bader Charge Analysis

Bader charge analysis was developed by Richard Bader. It is an intuitive way of dividing molecules into atoms with definition of an atom based purely on the electronic charge density 24 .

Zero flux surfaces on which the charge density is a minimum perpendicular to the surface were used to divide atoms. Typically in molecular systems, the charge density reaches a minimum between atoms where is a natural place to separate atoms from each other 24 .

Because the Bader analysis assumes that charge density maxima are located at the center of an atom, in the present calculations, the total charge was obtained by summing the core charge and the valance charge from the VASP code and the Bader analysis was done on this total charge density file.

After the calculation, we can obtain the electronic charges and atomic volumes of 30 atoms for each compound. To get the atomic charge for each atom, we use the valence electrons (for which the valence states were set by VASP calculation) minus the calculated electronic charge. The negative value of atomic charge indicates atom tends to gain electrons and the positive one indicates atom tends to lose electrons.

RESULTS AND DISCUSSION

In the present work, besides the size factor and the number of valence electrons (e), the total number of electron shells (s) is also considered to clarify the regularity of the atomic order of the sigma phase. We classify the A-B binary sigma systems involving transition elements according to the electron configuration of the constituent elements as shown in Fig. 1. The A-Al (A= Nb and Ta) systems, for which the main element Al is concerned, are classified into a separate category. The reason will be discussed in the following sections.

The systems with experimental site occupancies available are as follows:

1) SMe (atom A is with the smaller number of valence electrons than atom B) V-Mn (3d 3 4s 2 , 3d 5 4s 2 ), V-Fe (3d 3 4s 2 , 3d 6 4s 2 ), V-Co (3d 3 4s 2 , 3d 7 4s 2 ), V-Ni (3d 3 4s 2 , 3d 8 4s 2 ), Cr-Mn (3d 5 4s 1 , 3d 5 4s 2 ), Cr-Fe (3d 5 4s 1 , 3d 6 4s 2 ), Cr-Co (3d 5 4s 1 , 3d 7 4s 2 ) W-Re (5d 4 6s 2 , 5d 5 6s 2 );

2) LAs (atom A is with the larger total number of electron shells than atom B)

Re-Mn (5d 5 6s 2 , 3d 5 4s 2 );

3) LAs-LAe (atom A is with the larger total number of electron shells and the larger number of valence electrons than atom B)

Ru-Cr (4d 7 5s 1 , 3d 5 4s 1 ), Re-Cr (5d 5 6s 2 , 3d 5 4s 1 ), Re-V (5d 5 6s 2 , 3d 3 4s 2 ), Os-Cr (5d 6 6s 2 , 3d 5 4s 1 );

4) LAs-SMe (atom A is with the larger total number of electron shells and the smaller number of valence electrons than atom B)

Mo-Mn (4d 5 5s 1 , 3d 5 4s 2 ), Mo-Fe (4d 5 5s 1 , 3d 6 4s 2 ), Mo-Co (4d 5 5s 1 , 3d 7 4s 2 ), Ta-Rh (5d 3 6s 2 , 4d 8 5s 1 ), Ta-Pd (5d 3 6s 2 , 4d 10 ), W-Ru (5d 4 6s 2 , 4d 7 5s 1 ), Re-Fe (5d 5 6s 2 , 3d 6 4s 2 );

5) SMs-SMe (atom A is with the smaller total number of electron shells and the smaller number of valence electrons than atom B)

Nb-Re (4d 4 5s 1 , 5d 5 6s 2 ), Nb-Os (4d 4 5s 1 , 5d 6 6s 2 ), Nb-Ir (4d 4 5s 1 , 5d 7 6s 2 ), Nb-Pt (4d 4 5s 1 , 5d 9 6s 1 ), Mo-Re (4d 5 5s 1 , 5d 5 6s 2 ), Mo-Os (4d 5 5s 1 , 5d 6 6s 2 ), Mo-Ir (4d 5 5s 1 , 5d 7 6s 2 );

6) A-Al (A= Nb and Ta)

Nb-Al (4d 4 5s 1 , 3s 2 3p 1 ), Ta-Al (5d 3 6s 2 , 3s 2 3p 1 ).

Fig. 1 A-B (V(A)>V(B), where V(A) and V(B) are the molar volumes of element A and B in their hypothetic sigma phase structure from the CALPHAD assessments 18 respectively) binary sigma systems divided into different categories according to the electron configuration of the constituent elements. The right angled rectangle indicates no sigma phase existing or no experimental site occupancies available for the included systems; the rounded rectangle indicates experimental data for site occupancies are available for the included systems. Note that the existence of the sigma phase in Cr-Ni, Ta-V, Ti-Mn, Zr-Ir and Zr-Re systems is not conformed.

(LAs: Atom A is with the larger total number of electron shells than atom B; LAe: Atom A is with the larger number of valence electrons than atom B; SMs: Atom A is with the smaller total number of electron shells than atom B; SMe: Atom A is with the smaller number of valence electrons than The size factor has twofold meanings. The first is the size difference among crystal sites. Both CN and nearest neighbor (NN) sites affect the size difference of sites, which will be discussed in the following 3.1.1(a). The second is the atomic size difference between the constituent elements, which was discussed in Section 1. If only the size factor affecting atomic order is considered, it is obvious that among the two constituent elements, the one with a large atomic size preferentially occupy sites with large size.

V-Co V-Fe V-Mn V-Ni W-Re • ••• • Mo-Ru Mo-Tc Nb-Pd Nb-Rh Ta-Au Ta-Ir Ta-Os Ta-Pt Ta-Re • ••• • W-Ir W-
As respect to the atomic size sequence of the same element, normally, it follows the occupied site size sequence. However, it may not be always true owing to the influences of unlike NN atoms on atomic volume, which will be discussed in the following 3.1.1(b).

In the following discussion and shown in Fig. 2345, the atomic charge indicates the tendency of electron loss or gain. When the value is negative, it means the atom tends to gain the corresponding number of electrons; when the value is positive, the atom tends to lose electrons.

(a) Size difference among the five inequivalent sites

To clarify the size difference among the five inequivalent sites of the sigma phase, we calculated the atomic volume and the corresponding atomic charge of the two constituent elements in the complete set (2 5 =32) of ordered configurations (i.e. the stoichiometric end-member compounds in the following discussions) of the sigma phase for different binary systems. Fig. 2 presents the atomic volume varies with the atomic charge of the constituent elements in the 32 stoichiometric end-member compounds for 12 typical sigma phase systems. For systems in SMe, SMs-SMe categories and some systems (i.e. Ta-Pd and Ta-Rh systems) in LAs-SMe category, a nearly linear relationship with a constant slope can be observed between atomic volume and atomic charge for atoms on a specific site; for systems in LAs, LAs-LAe, A-Al (A= Nb and Ta) categories and a system (i.e. Mo-Mn system) in LAs-SMe category, no evident linear relationship can be observed as the atomic volume difference for a specific site when occupied by either A or B can be very large, even when the atomic charge is constant. The large difference is due to the influence of NN atoms which will be discussed in Section 3.1.1. (b). Fig. 3 shows the atomic volume versus atomic charge of the pure elements in the hypothetic sigma structure. In this respect, we can observe that the size difference among the five inequivalent sites without the effect of unlike NN atoms. If we consider a linear relation (with an almost constant slope) between atomic volume and atomic charge as observed in Fig. 2, and extrapolate to a specific atomic charge (e.g. the vertical line in Fig. 3), we can obtain the corresponding atomic volumes for all five sites. Then we can propose from Fig. 3 that the site size sequence is as follows:

4f (CN15) >8i 1(CN14) >8j (CN14) >8i 2(CN12) >2a (CN12)
. This result agrees with the weighted average of the interatomic distances reported for Cr-Fe sigma by Hall and Algie [1], i.e. 2.701 Å (4f)>2.652 Å (8i 1 )>2.638 Å (8j)>2.526 Å (8i 2 )>2.508 Å (2a).

The above results follow the analysis by considering crystal structure. Firstly, sites with large CN (i.e. CN=14 or 15) are expected to bear a large size, as with more surrounding atoms the space of a site is larger. Thus the size difference among different CN sites are expected to be 4f (CN15) > 8i 1(CN14) /8j (CN14) >8i 2(CN12) /2a (CN12) . Secondly, regarding the sites with the same CN, namely CN14 (i.e. 8i 1 and 8j) and CN12 (i.e. 8i 2 and 2a), we should consider the effect of the NN sites. The size of the site is expected to be larger when the surrounding small CN sites (i.e. CN=12) are fewer, which implies that the surrounding large CN sites are more. Table 1 shows the number of the NN sites for the five inequivalent sites of the sigma phase. The 8i 1 site is surrounded by 4 CN12 sites and the 8j site is surrounded by 5 CN12 sites, so the size of the 8i 1 site is expected to be larger than the 8j site. The 8i 2 site is surrounded by 2 CN12 sites and the 2a site is surrounded by 4 CN12 sites, so the size of the 8i 2 site is expected to be larger than the 2a site. The above analysis is well consistent with the calculation results based on Fig. 2 and Fig. 3, i.e. the site size sequence follows

4f (CN15) >8i 1(CN14) >8j (CN14) >8i 2(CN12) >2a (CN12) .
(b) Effect of unlike NN atoms and their configuration on atomic volume For some binary sigma compounds, the atomic volumes sometimes may not follow the size sequence of the correspondingly occupied site. To figure this out, the influencing factors on atomic volume are specially discussed. For a specific atom in the sigma phase structure, two factors determine its atomic volume. The first one is its occupying crystal site (i.e. one among 2a, 4f, 8i 1 , 8i 2 , 8j). The second concerns its NN atoms and their configuration or distribution around this atom.

The effect of the next NN atoms is assumed small that can be neglected. The NN atoms mainly affect its atomic charge and thus affect its atomic volume; the configuration of the NN atoms mainly affects the space to accommodate this specific atom (in this case the atomic charge changes in a small range) and thus affects its atomic volume.

It can be seen from Fig. 2 that for SMe and SMs-SMe categories, the atomic size and site size follow the same sequence, i.e. 4f (CN15) >8i 1(CN14) >8j (CN14) >8i 2(CN12) >2a (CN12) . Moreover, for the same or a small variation of the atomic charge, the atomic volume of the same element for a specific site is constant or changes but to a small extend. While, on the one hand, for LAs and A-Al (A=Nb and Ta) categories, for the same or a small variation of the atomic charge, the atomic volume difference of the same element for a specific site can be very large. It indicates that the configuration of the NN atoms have a large effect on atomic size for LAs and A-Al categories. On the other hand, the NN atoms can affect the atomic size relationship between different sites. Taking Re-Mn system (LAs) as an example, when Re occupy 8i 2 and 8j sites, and Mn occupy 8i 1 site, 4f and 2a sites occupied by Re or Mn respectively, the atomic sizes of Re on 8i 2 and 8j sites become close (normally the atomic size sequence of the same element follows the occupied site size sequence, i.e. 8j (CN14) >8i 2(CN12) ) as seen in, e.g. Fig. 4 (a). When Mn occupy 8i 1 and 8j sites, and in the meanwhile the number of the NN Re surrounding 8j site is large enough, the atomic sizes of Mn on 8i 1 and 8j follows 8j>8i 1 (normally the atomic size sequence of the same element follows the occupied site size sequence, i.e. 8i 1(CN14) >8j (CN14) ) as seen in, e.g. Fig. 4 (b). Some interesting phenomena found in Fig. 2 can also be explained by considering the effects of NN atoms, which further validate our arguments. Fig. 5 (a-f) shows the calculated atomic volume varies with the atomic charge of W and Re in 6 stoichiometric sigma end-member compounds for the W-Re system. By replacing site by site W atom by Re atom, one obtains 6 compounds in succession going from pure W sigma to pure Re sigma. By following the path W→2a→4f→8i 1 → 8i 2 →8j→Re, the atomic charge changes in a way that i) the pure sigma compounds show a smaller range of atomic charge around zero in comparison with binary ones; ii) when the content of Re increases, in general the atomic charge of both W and Re increases. For W-Re system, Re atoms tend to gain electrons and W atoms tend to lose electrons. The above conclusions are universal and valid for all the binary sigma systems. As a corollary with the atomic charge increasing, for the corresponding compound, the content of atoms which tend to gain electrons increases. Notably, it can be seen from Fig. 2 that for SMe and SMs-SMe categories, atom B tend to gain electrons; for LAs-LAe and LAs categories, atom A tend to gain electrons. With all the above observations, we can explain the following phenomenon: for SMe and SMs-SMe category, when the atomic charge increases, the size difference among the different CN sites (CN12, CN14 and CN15) decreases.

That is because when the atomic charge increases, the content of atom B increases, and thus irrespective of the atom, the number of NN atom B (smaller than atom A) increases. However, this observation is not obvious for LAs-LAe and LAs categories, as the configuration of the NN atoms affect atomic volume remarkably.

3.1.2. Electronic factors: valence electron and electron shell Fig. 2 indicates that when the atomic charge decreases, the atomic volume increases. Moreover, it also indicates that between the two constituent elements, the one with smaller total number of electron shells or smaller number of valence electrons tends to lose electrons; the one with larger total number of electron shells or larger number of valence electrons tends to gain electrons. On the other hand, Fig. 3 indicates that generally, atoms on 2a and 8i 2 sites tend to gain electrons and atoms on 4f, 8i 1 and 8j sites tend to lose electrons. It is worth mentioning that the atoms on 8j site bear a relatively larger atomic charge range as compared to the atoms on the other sites. In some cases, atoms on 8j site can either gain or lose electrons. Thus in general, between the two constituent elements, the one with smaller total number of electron shells or smaller number of valence electrons prefers occupying large CN sites (4f, 8i 1 , 8j) and the one with larger total number of electron shells or larger number of valence electrons prefers occupying small CN sites (2a, 8i 2 ).

The above argument about the valence electron factor is consistent with the previous investigations 1-2, 26 that atoms poor in d-electrons preferentially occupy sites with large CN (4f, 8i 1 , 8j) and atoms rich in d-electrons preferentially occupy sites with small CN (2a, 8i 2 ). However, they 1-2, 26 did not consider the effect of the total number of electron shells on atomic order, which makes it unable to well explain the experimental site occupancies. Besides, Berne [START_REF] Berne | Ab Initio Study of Metastability in Refractory Metal Based Systems[END_REF] indicates that CN12 sites show predominance for atoms with filled or nearly filled or else empty d shells. The reason is that the CN12 sites have approximate icosahedral symmetry causing high degeneracy of electronic d-like levels [START_REF] Sluiter | Site occupation in the Cr-Ru and Cr-Os σ phases[END_REF][START_REF] Berne | Ab Initio Study of Metastability in Refractory Metal Based Systems[END_REF] . Atoms with half-filled d shells would have high density of states when occupying the CN12 site and thus consequently occupy high CN sites, which allows a better separation of bonding and antibonding d bands 5 .

For A-Al (A= Nb and Ta) category shown in Fig. 2 (k) (l), the characteristics of atomic charge seem a little different, i.e. the tendency of electron loss or gain for the two constituent elements is similar. Hence for this category, the atomic order is mainly determined by the size factor, i.e. large atoms (atom A) occupy large CN (4f, 8i 1 , 8j) sites and small atoms (atom B) occupy small CN (2a and 8i 2 ) sites. This is consistent with the measured occupancies presented in Section 3.2.3.

The A-Al (A= Nb and Ta) systems could wrongly be classified into the LAs-LAe category as atoms Nb and Ta bear both a larger number of valance electrons and larger total number of electron shells than atom Al. However, the behavior of A-Al category as mentioned above is similar to that of LAs-SMe category. The compromise of LAs and SMe factors causes the tendency of electrons loss or gain for the two constituent elements similar. Actually, as can be seen in Fig. 2 (f) (k) (l), the behavior of A-Al (A= Nb and Ta) systems is similar as that of Mo-Mn system (which belongs to LAs-SMe category). This is consistent with the discovery by Joubert 2 , who indicates that Al behaves as if it had 9 or 10 valence electrons in the sigma phase structure. Apparently, the behavior of A-Al category in Fig. 2 (k) (l) results from a compromise between the effects of LAs and SMe factors. Besides, Fig. 2 (k) (l) shows that Ta atoms prefer to gain electrons as compared to Nb, because Ta bears a larger total number of electron shells than Nb. Thus LAs factor plays more important role for Ta-Al system. ) and the one with the larger total number of electron shells or the larger number of valence electrons prefer occupying small CN sites (i.e. 2a, 8i 2 ). To reveal the atomic order among the large CN sites (i.e. 4f, 8i 1 , 8j) and between the small CN sites (i.e. 2a, 8i 2 ) of the sigma phase, we present in Fig. 6 the measured site occupancies from the literature category by category and considering the accuracy, generally, recent data were collected. Fig. 7 shows the size differences of the constituent elements of the binary sigma phase with respect to their differences in the number of valence electrons and the total number of electron shells. For a specific system, when the size difference is smaller and in the meantime, the differences in both the number of valence electrons and the total number of electron shells are larger as compared to other systems, the electronic factors affect the atomic order more remarkably.

It should be noted that the experimental site occupancies are results of competition and cooperation of these factors. In this section, we aim in dissociating the effects of individual influencing factor on atomic order based on first-principles calculation results in Section 3.1 and experimental data from the literature in order to further analyze and explain the atomic order.

Effect of the number of valence electrons on atomic order

Fig. 6 (a) shows that in most cases for the SMe category, the site occupancy follows Y 2a (A)> Y 8i2 (A) and Y 4f (A)>Y 8j (A)>Y 8i1 (A). We showed above that the size sequence of the crystal site is 4f>8i 1 >8j>8i 2 >2a. It is thus clear that it is the electronic factor (SMe) that affects the sequence of atomic order to be 2a>8i 2 and 8j>8i 1 .

Besides, for all the measurements in Fig. 6 (a), only among the measurements of Cr-Co, Cr-Mn and V-Ni systems, there are experimental data indicating that 4f site is not the most favorable site for atom A, which exhibit Y 8i1 (A)>Y 4f (A) and Y 8j (A)>Y 4f (A) for Cr-Co system, Y 8j (A)>Y 4f (A)

for Cr-Mn and V-Ni systems, respectively. It must be owing to the effect of the electronic factor (SMe), since in terms of the size factor, 4f site should be the most favorable site for atom A. It can also be seen from Fig. 7 (a) that the electronic factor can be more remarkable for these systems than the other ones in SMe category, as for Cr-Mn system, the two constituent atoms bear the smallest size difference, for V-Ni system, the two constituent atoms bear the largest difference of valence electrons, and Cr-Co system is in between.

Therefore, we can expect that SMe factor affects the atomic order in a sequence of 8j(A)> 8i 1 (A)>4f(A)>2a(A)>8i 2 (A). Vice versa, LAe factor affects the atomic order in a sequence of 8i 2 (A)>2a(A)>4f(A)>8i 1 (A)>8j(A).

Effect of the total number of electron shells on atomic order

For LAs category presented in Fig. 6 (b), only measured site occupancies for Re-Mn system is available. The figure shows Y 8i2 (A)>Y 2a (A) and Y 8i1 (A)>Y 8j (A). Moreover, the difference in A occupancies in 8i 2 and 2a sites, and in 8i 1 and 8j sites for LAs category, is too large to explain with only size factor. It is expected that LAs factor affects the atomic order in a sequence of 8i 2 (A)> 2a(A) and 8i 1 (A)>8j(A).

On the other hand, for LAs-LAe category, Fig. 7 (b) indicates that the electrons factors affect the atomic order most remarkably for the Re-V system. Fig. 6 (d) shows for Re-V system, the site

occupancies follows Y 8i2 (A) > Y 2a (A) > Y 8i1 (A) > Y 4f (A) > Y 8j (A)
and the difference in A occupancies on 8i 1 and 8j sites is dramatically large. Thus we can expect the electronic factors (both LAs and LAe) affect the atomic order in a sequence of 8i 2 >2a>8i 1 >4f/8j for LAs-LAe category. As discussed above, for LAe and LAs-LAe categories the electronic factors affect the atomic order in a sequence of 8i 2 >2a>4f>8i 1 >8j and 8i 2 >2a>8i 1 >4f/8j, respectively. In this regard, LAs factor must affect the atomic order in a sequence of 8i 1 >4f. Thus LAs factor affects the atomic order in a sequence of 8i 2 >2a>8i 1 >4f/8j. Vice versa, SMs factor affects the atomic order in a sequence of 4f/8j>8i 1 >2a>8i 2 .

As mentioned in Section 1, Spooner and Wilson 8 indicated that the site occupancies of 2a, 4f, and 8i 2 sites were mainly determined by size factor and the site occupancies of 8i 1 and 8j sites were in addition determined by valence electrons. The reason for Spooner and Wilson 8 reaching the above conclusion is that for some systems, the effects of the size and electronic factors are similar, such as SMe and SMs-SMe categories for which both size and electronic factors cause the atomic order in a sequence of 4f/8i 1 /8j>8i 2 /2a. Actually, there are many exceptions considering this argument. One is that for Cr-Mn, Cr-Co and V-Ni systems in SMe category, 4f site is not the most favorable site for atom A while the size factor indicates otherwise. Another one is that for Re-V system in LAs-LAe category, 8i 2 and 2a sites have become the most favorable sites for atom A, because of the larger effects of the electronic factors (LAs and LAe) than the size factor. The influencing factors on atomic order for this category are the size and SMe factors which lead to a sequence of 4f>8i 1 >8j>8i 2 >2a and 8j>8i 1 >4f>2a>8i 2 , respectively. In this category, the site occupancy data on sites with the same CN (CN12: 8i 2 vs 2a, CN14: 8i 1 vs 8j) is close. Most data show Y 2a (A)>Y 8i2 (A) and Y 8j (A)>Y 8i1 (A). This is because SMe factor affects the atomic order more remarkably than the size factor.

For Cr-Mn, Cr-Co and V-Ni systems, as mentioned in Section 3.2.1, the effect of SMe factor on atomic order is particularly notable which leads to Y 8j (A)/Y 8i1 (A)>Y 4f (A) or Y 8j (A)>Y 4f (A). For the other systems, the size factor determines the site occupancy on 4f site. It is worth mentioning that data for Cr-Co system from Cieślak [START_REF] Cieślak | Site oiccupancy and lattice parameters in sigma-phase Co-Cr alloys[END_REF] and data for W-Re system from Berne [START_REF] Berne | Site occupancy in the Re-W sigma phase[END_REF] show that site occupancies is inverted among sites with the same CN (2a vs 8i 2 ; 8i 1 vs 8j). This is due to a competition between electronic and size factors.

For V-Ni system, Joubert 2 also measured the site occupancies by using the XRD method, but the data are not accurate due to the weak XRD contrast between the two constituent elements, which were not considered in the present work. On the contrary, the early data measured by Kasper 6 are more accurate, since they conducted neutron diffraction (ND) experiments yielding a better resolution for site occupancy measurements.

(b) LAs category (Fig. 6 (b))

The influencing factors on the atomic order for this category are the size and LAs factors which lead to a sequence of 4f>8i 1 >8j>8i 2 >2a and 8i 2 >2a>8i 1 >4f/8j, respectively.

For the LAs category, experimental data are available for Re-Mn system only. Both size and electronic factors affect the atomic order in a sequence of 8i 2 >2a and 8i 1 >8j. The size factor determines the site occupancy of 4f site.

(c) LAs-SMe category (Fig. 6 (c))

The influencing factors on the site occupancy for this category are the size, LAs and SMe factors which lead to a sequence of 4f>8i 1 >8j>8i 2 >2a, 8i 2 >2a>8i 1 >4f/8j and 8j>8i 1 >4f>2a>

8i 2 , respectively. The atomic order results from a competition among the three factors above. Atom A prefers occupying large CN sites (i.e. 4f, 8i 1 , 8j) rather than small CN sites (i.e. 2a, 8i 2 ), because of the both size and SMe factors.

For Mo-Fe, Mo-Mn and Re-Fe systems, Fig. 6 (c) presents Y 8i1 (A)>Y 8j (A) and Y 8i2 (A)>Y 2a (A), because the effect of SMe factor on atomic order is less significant than the combined effects of LAs and size factors. Besides, the difference of site occupancies between 8i 1 and 8j sites for Re-Fe system is the largest among the three systems. It is because of the effect of LAs factor, as for Re-Fe system the size difference between the two constituent elements is the smallest and n (n=s(A)-s(B)=2) is the largest among the three systems as shown in Fig. 7(c).

From Mo-Co to Ta-Rh system, SMe factor increasingly affects the atomic order as indicated in In addition, it can be seen from Fig. 7 (c) that the value of n (n=s(A)-s(B)) and e(A)-e(B) for W-Ru system is the same as that for Mo-Fe system, but the site occupancy orderings for these two systems are different as presented in Fig. 6 (c). The reason is that the size difference between W and Ru atoms is far smaller than that for Mo-Fe system (see Fig. 7(c)), thus less remarkable effect of size factor on the atomic order of W-Ru system than that of Mo-Fe system.

(d) LAs-LAe category (Fig. 6 (d))

The influencing factors on the atomic order for this category are the size, LAs and LAe factors leading to a sequence of 4f>8i 1 >8j>8i 2 >2a, 8i 2 >2a>8i 1 >4f/8j and 8i 2 >2a>4f>8i 1 >8j, respectively. The atomic order results from a competition among the three factors above.

The site occupancy behavior of Os-Cr, Re-V and Ru-Cr systems is almost completely disordered 2 . This is because of the equivalent effects of the size and electron factors. A less disordered state for Re-Cr system as compared to Os-Cr, Re-V and Ru-Cr systems is because of a larger effect of the size factor.

For Re-V system, the size difference between the two constituent elements is the smallest and the value of n (n=s(A)-s(B)) and e(A)-e(B) is the largest (see Fig. 7 (c)), which indicates that the effects of electron factors are stronger for Re-V system as compared to other systems. The experimental site occupancy of Re

-V system shows Y 8i2 (A)>Y 2a (A)>Y 8i1 (A)>Y 4f (A)>Y 8j (A),
which is an evidence that the electronic factors affect more dramatically the atomic order than the size factor.

It is worth noting that as mentioned in Section 1, Sluiter and Paturel 17 clarified that for Ru-Cr and Os-Cr systems, all five crystal sites were equivalent as no overwhelming ordering preference of constituent elements. However, the five crystal sites are different in CN and NN sites. With the findings discussed in Section 3.2, we can conclude that the reason for no overwhelming ordering preference is because of a competition among the effects of electronic and size factors rather than an equivalence of five crystal sites.

(e) SMs-SMe category (Fig. 6 (e))

The influencing factors on the atomic order for this category are the size, SMs and SMe factors which lead to a sequence of 4f>8i 1 >8j>8i 2 >2a, 4f/8j>8i 1 >2a>8i2 and 8j>8i 1 >4f>2a> 8i 2 , respectively.

For Mo-Re system, in general, Faradfar's 30 measurements show Y 8i2 (A)>Y 2a (A). This is caused by the size factor. Even though the size difference between Mo and Re atoms is the smallest among all systems in SMs-SMe category, it is the small value of n (n=s(A)-s(B)) and e(A)-e(B) that causes the size factor more prominent (see Fig. 7 (d)).

(f) A-Al (A= Nb and Ta) category (Fig. 6 (f))

As mentioned in Section 3.1.3, A-Al category behaves like LAs-SMe category with regard to the electron loss or gain. Thus the influencing factors on atomic order for this category are the size, LAs and SMe factors which lead to a sequence of 4f>8i 1 >8j>8i 2 >2a, 8i 2 >2a>8i 1 >4f/8j and 8j>8i 1 >4f>2a>8i 2 , respectively.

Fig. 6 (f) shows that in general, atom A and Al occupy large CN (i.e. 4f, 8i 1 , 8j) sites and small CN (2a and 8i 2 ) sites, respectively. In addition, the site occupancy is inverted between sites with the same CN (2a and 8i 2 ; 8i 1 and 8j). The above observations are because of a compromise among the three influencing factors. Besides, the measurements present that Y 8i1 (A)/Y 8j (A)>Y 4f (A), which is caused by the effects of both LAs and SMe factors.

Discussion

In view of the size and electron configuration of the constituent elements, we have classified all the binary sigma phase systems into different categories. With this classification approach, it is possible to study the tendency of electrons loss or gain of the constituent elements and obtain the regularity of atomic order as investigated in the present work

The present findings can not only explain various results for stable sigma phase but also the calculation results for hypothetically unstable sigma phase. As mentioned in Section 1, Crivello et al. 5 calculated the site occupancies of the sigma phase in binary Re-X (X= Co, Cr, Fe, Hf, Mn, Mo, Nb, Ni, Os, Ru, Ta, Tc, Ti, V, W and Zr) systems including stable sigma compounds (Re-V, Re-Cr, Re-Mn, Re-Fe, Nb-Re, Mo-Re, Ta-Re and W-Re) as well as some hypothetically unstable ones (Ti-Re, Zr-Re, Hf-Re, Re-Co, Re-Ni, Re-Tc, Re-Ru and Re-Os). Some calculation results cannot be well explained in that work as already mentioned in Section 1 but are interpretable by using the analysis method in the present work as follows.

Among the sigma phase studied by Crivello et al. indicated that site occupancy behavior for the sites with the same CN (i.e. 2a vs 8i 2 ; 8i 1 vs 8j) is different. Taking Re-Os vs Re-Tc and Re-V systems as an example, for Re-Os (belongs to SMe category) system, the site occupancies present the order of 8j>8i 1 which is because of the effect of SMe factor. For Re-Tc (belongs to LAs) and Re-V (belongs to LAs-LAe) systems, the site occupancies present the order of 8i 1 >8j which is because of the effects of size, LAs and LAe factors.

In addition, we are also expecting to use the present method to investigate the atomic order for other non-stoichiometric intermetallic compounds, like Chi phase and Mu phase, etc.

CONCLUSIONS

The experimental site occupancies are results of competition and cooperation of the size factor and electronic factors (i.e. the number of valence electrons and the total number of electron shells).

The present work dissociated the effect of individual influencing factor on the atomic order for the binary sigma phase. All accurately measured site occupancies can be well explained by using the following conclusions.

1. Both the coordination number (CN) and nearest neighbor crystal sites affect the size of the five crystal sites of the sigma phase. The size of the five inequivalent crystal sites of the sigma phase follows 4f (CN15) >8i 1(CN14) >8j (CN14) >8i 2(CN12) >2a (CN12) .

2. In the binary sigma compounds, between the two constituent elements, the one with a smaller total number of electron shells or smaller number of valence electrons tend to lose electrons and thus the atomic size decreases; the one with a larger total number of electron shells or larger number of valence electrons tend to gain electrons and thus the atomic size increases. On the other Mn) from Elliott [START_REF] Elliott | Structure of the phase TiMn ang the Indexing of Powder patterns of sigam type phases[END_REF] is available. The predicted molar volume of the Mn-Ti sigma compound is much higher than the measurements, which is presented in

  atom B.) 3.1. Size and electronic factors affecting atomic order 3.1.1. Size factor s(A)<s(

Fig. 6 .Fig. 7 .

 67 Fig. 6. Site occupancy, Ys(A), varies with mole fraction of A, X(A). Measured data are from Ref.

Fig. 7

 7 Fig. 7 (c). For Mo-Co system, Fig. 6 (c) shows Y 4f (A)>Y 8i1 (A)≈Y 8j (A) and Y 8i2 (A)>Y 2a (A). It is

5

  Hf-Re, Ta-Re, W-Re and Re-Os systems belong to SMe category and Ti-Re, Zr-Re, Nb-Re and Mo-Re systems belong to SMs-SMe category. For SMe and SMs-SMe categories, both size and electronic factors cause the atomic order follow 4f/8i 1 /8j>8i 2 /2a, that is why the site occupancies of Nb-Re, Mo-Re, Ta-Re, W-Re, Ti-Re, Zr-Re and Hf-Re systems behave more-ordered 5 . As respect to Re-Os system, the site occupancies present relative less-ordered at the finite temperature of 2000K (see Fig.9in Ref. 5), which is because that the effects of the size and electronic factors are both small for this system.Re-Mn and Re-Tc systems belong to LAs category, while Re-V and Re-Cr belong to LAs-LAe category and Re-Fe, Re-Co, Re-Ni and Re-Ru belong to LAs-SMe category. A competition of the influencing factors makes a less-ordered state in these categories. Besides,Crivello et al. 5

  Fig. A. 1. Calculated molar volumes of Mn-X (X=Cr, Mo, Re, Tc, Ti, V) sigma phase compared

  order (i.e. atomic constituent distribution or site occupancy preference on inequivalent sites of a crystal structure) of the sigma phase is driven by both size and electronic (the number of valence electrons and the total number of electron shells) factors. By classifying the binary sigma systems into different categories according to the size and electron configuration of the two constituent elements, the site occupancy regularity of the sigma phase can be observed and analyzed.

Table 1 .

 1 Number of nearest neighbor (NN) atoms for the five inequivalent sites of the sigma phase Atomic volume with respect to atomic charge of the constituent elements in 32 (2 5 =32) stoichiometric sigma end-member compounds (e.g. Mo 2 Mo 4 Mo 8 Mo 8 Mo 8 , Re 2 Mo 4 Mo 8 Mo 8 Mo 8 etc.) for binary systems calculated using VASP and Bader Charge Analysis. (Every figure contains Re 4 Mn 8 Re 8 Re 8 ; b) Re 2 Re 4 Mn 8 Re 8 Mn 8 . Every figure contains 30 points but some of them are overlapped.

	16

Atomic Charge / eRe-Mn Mn (j)

Atomic Charge / e W-Re

hand, atoms on 2a and 8i 2 sites tend to gain electrons and those on 4f, 8i 1 and 8j sites tend to lose electrons. Hence between the two constituent elements, the one with a larger size, smaller number of valence electrons or smaller total number of electron shells prefer occupying large CN sites (i.e. 4f, 8i 1 , 8j) and the one with a smaller size, larger number of valence electrons or larger total number of electron shells preferring occupy small CN sites (i.e. 2a, 8i 2 ). The site occupancy for a specific system is a compromise of the three influencing factors.

3. The dissociated effect of the electronic factors leads to the following atomic order: LAs: 8i2>2a>8i1>4f/8j; SMs: 4f/8j>8i1>2a>8i2; LAe: 8i2>2a>4f>8i1>8j; SMe: 8j> 8i1>4f>2a>8i2.

APPENDIX A: MOLAR VOLUME ASSESSMENTS FOR THE SIGMA PHASE IN THE MN-X (X=CR, MO, RE, TC, TI, V) BINARY SYSTEMS By using the CALPHAD approach, Liu et al [START_REF] Liu | Modeling of molar volume of the sigma phase involving transition elements[END_REF] have assessed the molar volume of the sigma phase at room temperature and atmospheric pressure for binary systems concerning 20 elements Al, Au, Co, Cr, Fe, Ir, Mo, Nb, Ni, Os, Pd, Pt, Re, Rh, Ru, Ta, Tc, V, W and Zr. As a supplement of that work, we assessed Mn-X (X=Cr, Mo, Re, Tc, Ti, V) sigma binary systems by using the same model (for details see Ref. m 3 /mol. The agreements between different data are fairly good except for Mn-Ti system. Only measurements with molar volume of 7.19×10 -6 m 3 /mol (a=8.88 Å, c=4.54 Å, at about 48.6 at.%


C Desbiez

M-L Domingo Calap

M Pitrat

C Wipf-Scheibel

G Girardot

I Ferriol

J- J Lopez-Moya

H Lecoq

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Determinism of virulence of cucumber vein yellowing virus (CVYV) in melon and insights into the durability of resistance

Cecile Desbiez, Maria Luisa Domingo Calap, Michel Pitrat, Catherine

Wipf-Scheibel, Gregory Girardot, Inmaculada Ferriol, Juan Jose Lopez-Moya,

Hervé Lecoq

To cite this version: Genetic resistance is a sustainable way of controlling plant viruses, but it requires the presence of resistance genes in the host germplasm. Besides, its durability can be reduced by resistancebreaking isolates, either pre-existing in the environment or emerging after the deployment of the resistance. In the case of cucumber vein yellowing virus (CVYV, genus Ipomovirus), an emerging virus on cucurbits in the Mediterranean Basin, few resistances are available in melon.

The accession PI 164323 was found to display complete resistance to the strain CVYV-Esp, and accession HSD 2458 presented a tolerance, i.e. very mild symptoms in spite of virus accumulation in inoculated plants. The resistance is controlled by one dominant allele Cvy-1 1 , while the tolerance is controlled by a recessive allele cvy-2 independent from Cvy-1 1 . Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be strain-specific since many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaics on PI 164323 was obtained. It differed from the parental strain by a single amino-acid change in the VPg-coding region. The effect on the mutation was confirmed by reverse genetics using a CVYV infectious clone. Competition experiments suggested a fitness cost of the resistancebreaking mutation in susceptible melon. Our results highlight the need to combine cultural practices and/or additional genes to develop a more durable control of CVYV than the use of the Cvy-1 1 allele alone.


Nafees Bacha

Huy Phong Dao

Ali Atoui

Florence Mathieu

John O'callaghan

Olivier Puel

Thierry Liboz

Alan D W Dobson

Ahmed Lebrihi
email: lebrihi@ensat.fr

Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in Aspergillus westerdijkiae

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Aspergillus westerdijkiae is a producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone [START_REF] Atoui | Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase gene in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A[END_REF][START_REF] Balcells | Effect of fungal metabolites and some derivatives against Tribolium castaneum (Herbst) and Nezara viridula (L.)[END_REF][START_REF] Gaucher | Isolation of orsellinic acid synthase[END_REF][START_REF] Torres | Bactericidal and fungicidal activity of Aspergillus ochraceus metabolites and some derivatives[END_REF]. These two metabolites belong to the partially reduced or methylsalicylic acid (MSA) type polyketide group and have same chemical structure but are different stereochemically. Isoasperlactone and asperlactone are on one hand efficient antimicrobial agents [START_REF] Rosenbrook | New metabolite from an unidentified Aspergillus species[END_REF][START_REF] Torres | Bactericidal and fungicidal activity of Aspergillus ochraceus metabolites and some derivatives[END_REF], and on the other hand they contain ovicidal activities against Nezara viridula [START_REF] Balcells | Effect of fungal metabolites and some derivatives against Tribolium castaneum (Herbst) and Nezara viridula (L.)[END_REF][START_REF] Balcells | Bactericidal and fungicidal activity of Aspergillus westerdijkiae metabolites and some derivatives[END_REF]. It has been reported that asperlactone and isoasperlactone presented a strong anti-bacterial and anti-fungal activities when compared with other antimicrobial agents [START_REF] Balcells | Bactericidal and fungicidal activity of Aspergillus westerdijkiae metabolites and some derivatives[END_REF].

It has been previously demonstrated that during the biosynthesis of MSA type polyketides, the first step is the formation of 6-methylsalicylic acid (6-MSA) by the condensation of one molecule of acetyl-coA and three molecules of malonyl-coA [START_REF] David | Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2)[END_REF]. This reaction is catalyzed by a multifunctional enzyme system known as methylsalicylic acid synthase (MSAS). This enzyme has been reported to catalyze several MSA type polyketide compounds like patuline in Penicillium patulum [START_REF] Beck | The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases[END_REF] and Byssochlamys fulva [START_REF] Puel | The inability of Byssochlamys fulva to produce patulin is related to absence of 6-methylsalicylic acid synthase and isoepoxydon dehydrogenase genes[END_REF] and avilamycin in Streptomyces viridochromogenes [START_REF] Gaisser | Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57[END_REF].

Not much is known about the biosynthetic pathways of isoasperlactone and asperlactone, except for a hypothetical scheme proposed by [START_REF] James | 17O NMR in biosynthetic studies: aspyrone, asperlactone and isoasperlactone, metabolites of Aspergillus melleus[END_REF]. According to this scheme, the isomeric metabolites aspyrone, isoasperlactone and asperlactone are derived from a common biosynthetic precursor, the diepoxide. [START_REF] James | 17O NMR in biosynthetic studies: aspyrone, asperlactone and isoasperlactone, metabolites of Aspergillus melleus[END_REF] further stated that asperlactone is formed directly from the diepoxide, while isoasperlactone is formed from diepoxide via aspyrone pathway (Fig. 1).

Many PKS genes have been sequenced in filamentous fungi including A. fumigatus and A. niger [START_REF] Metz | Production of polyunsaturated fatty acids by polyketide synthase in both prokaryotes and eukaryotes[END_REF][START_REF] Niermanm | Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus[END_REF][START_REF] Pel | Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88[END_REF][START_REF] Sebastian | Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans[END_REF]. It is therefore possible to use the most conserved region among these PKS genes (Fig. 2) as primers for a PCR based cloning strategy. For this purpose, mainly the ketosynthase (KS) and acyle transferase (AT) domains, i.e. the most conserved domains among different PKSs (Fig. 2), have been utilized. The previously designed pairs of degenerated primers KS1/KS2, LC1/LC2c and LC3/LC5c [START_REF] Bingle | Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes[END_REF][START_REF] Nicholson | Design and utility of oligonucleotide gene probes for fungal polyketide synthases[END_REF] has been already utilized in our laboratory to clone eight KS domains from different PKSs [START_REF] Atoui | Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase gene in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A[END_REF], including one from MSAS in A. westerdijkiae. Recently one of these PKS genes has been found to be involved in the biosynthesis of ochratoxine A [START_REF] Bacha | Aspergillus westerdijkiae polyketide synthase gene ''aoks1" is involved in the biosynthesis of ochratoxin A[END_REF].

In this paper, we report sequencing and functional characterization of the first MSAS-type PKS gene ''aomsas" identified in A. westerdijkiae. It is involved in the biosynthesis of two pharmacologically important lactonic metabolites, i.e. isoasperlactone and asperlactone. We also demonstrate that 6-methylsalicylic acid (6-MSA), aspyrone and diepoxide are intermediates in their biosynthetic pathway.

Materials and methods

Fungal strain and culture conditions

Aspergillus westerdijkiae NRRL 3174 strain was grown for sporulation at 25 °C on potato dextrose agar for 7 days. Spores were collected using a solution of 0.01% (v/v) Tween 80, counted by using Thoma Bright line counting chamber (Optick labor), and stored at À20 °C in 25% (v/v) glycerol before use. Conidia were inoculated (density $10 6 /mL) into 250 mL Erlenmeyer flasks containing 100 mL synthetic medium (SAM) at 25 °C for 2-18 days, without shaking. The composition of SAM (per liter of distilled water) was: 3 g NH 4 NO 3 , 26 g K 2 HPO 4 , 1 g KCl, 1 g MgSO O), and 50 g glucose. The pH of the medium was adjusted to 6.5 by the addition of 2 N HCl. Mycelium was harvested by filtration through a 0.45-lM filter, grounded in liquid nitrogen and then stored at À80 °C before nucleic acid extraction. Secondary metabolites were extracted from filtrates of 2-18 days old cultures medium. Three replications of each sample were analyzed.

Nucleic acid extraction

Rapid method of genomic DNA extraction [START_REF] Lui | Rapid mini preparation of fungal DNA for PCR[END_REF] was used for transformants screening by PCR. Large quantity genomic DNA was extracted by CTAB extraction method [START_REF] Gardes | ITS primers with enhanced specificity for basidiomycetes -application to the identification of mycorrhizae and rusts[END_REF]. The quality and quantity of DNA were estimated by measuring OD 260 nm/OD 280 nm and OD 260 nm, respectively.

Total RNA was extracted from A. westerdijkiae using the Tri-reagent (Euromedex France) DNA/RNA/Protein extraction kit. The quality and quantity of RNA was checked by the OD 260 nm/OD 280 nm ratio and agarose gel electrophoresis according to standard protocols [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF].

Cloning of aomsas gene in A. westerdijkiae

Degenarated primers based Gene walking approach and RACE-PCR was used to clone aomsas gene in A. westerdijkiae. Fig. 2 marks positions while Table 1 lists all the primers used during this study. The degenerated primer pair LC3/LC5c [START_REF] Bingle | Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes[END_REF] designed for ketosynthase (KS) domains of MSAS-type PKSs was used to isolate putative PKS gene fragment of 700 bp [START_REF] Atoui | Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase gene in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A[END_REF]. The degenerated primers MS2 and MS4 targeting the conserved regions GVSAMGFPW and GVVHAAGV were further used with specific primer MS1 allowed by PCR to clone an additional 2 kb of the gene. Using the same strategy, an additional 1.3 kb downstream sequence of the aomsas gene was subsequently cloned. Finally the 5 0 and 3 0 extremity of the aomsas gene were subsequently amplified by using a RACE-PCR approach on cDNA (kit supplied by Invitrogen with 5 0 -primer and 3 0 -primer). Each time the PCR products obtained were cloned in pCR2.1-Topo vector (Invitrogen) and sequenced. Alignments of the sequenced fragments were performed to search for consensus.

PCR and sequencing

PCR was performed with the Taq recombinant polymerase (Invitrogen, USA). Amplification was carried out in a 50-lL reaction mixture containing: 5 lL of Taq polymerase 10Â buffer, 1.5 lL of 50 mM MgCl 2 , 1 lL of dNTP 10 mM of each (Promega), 1 lM of each primer, 1.5 U of Taq, about 200 ng of DNA genomic, H 2 O up to 50 lL. Reaction conditions were: 94 °C for 4 min, (94 °C for 45 s, 53 °C for 45 s and 72 °C for 1 min) Â 30 cycles followed by an incubation at 72 °C for 10 min. The amplified products were examined by 1% (w/v) agarose gel. The PCR products were cloned into pCR2.1-Topo vector according to the supplier's instructions. Sequencing of the fragments was performed by Genomexpress (Grenoble, France).

Data analysis

The deduced amino acid sequence was determined using the http://www.expasy.org/tools/dna.html site while protein-protein Blast (Blastp) searches were conducted at the GenBank database: http://www.ncbi.nlm.nih.gov. The alignments were conducted using the website http://prodes.toulouse.inra.fr/multalin/multalin. html. 1) used in gene walking experiment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Reverse transcription PCR

The expression of aomsas gene in A. westerdijkiae was examined by using reverse transcription PCR (RT-PCR) with two specific primers AoLC35-2L and AoLC35-2R designed from the KS domain of aomsas gene (Table 1). For RT-PCR, total RNA was treated with DNase I (Promega) to remove DNA contamination. cDNA was synthesized from each sample with Advantage RT-for-PCR Kit (BD Biosciences) according to the supplier's manual. cDNA amplification were performed using Taq recombinant polymerase (Invitrogen, USA). Beta tubulin was used as positive control using primers TubF and TubR (Table 1).

Plasmid construction and transformation

To construct TopoMShph, the aomsas gene replacement vector, a 1995 bp amplified fragment containing SalI restriction site was obtained using MSdF and MSdR primers (Fig. 4a and Table 1). This fragment was ligated into pCR2.1-Topo plasmid generating the TopoMS plasmid. TopoMS and pID2.1 plasmids were restricted using SalI enzyme. The hph cassette (2.4 kb) obtained from pID2.1 was ligated with the restricted TopoMS plasmid to produce TopoMShph vector, in which the 5 0 and 3 0 -ends of hph cassette are flanked by aomsas gene fragments. To construct the complementation vector pAN-MSAS, PCR primers MSdF1 and MSdR1 were designed to amplify a 5-kb aomsas gene fragment. The amplified fragment was cloned in pAN8-1 plasmid containing phleomycine resistance cassette.

The aomsas gene inactivation and complementary mutant production were achieved by preparing protoplasts of the corresponding strains and transforming with TopoMShph (Fig. 3b) and pAN-MSAS, respectively, as previously described [START_REF] Jaoanne | Regulation and role of the STE12-like transcription factor from the plant pathogen Colletotrichum lindemuthianum[END_REF][START_REF] O'callaghan | A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus[END_REF]. Lysing enzyme (40 mg/mL) (Sigma) was used for the preparation of protoplasts.

Screening of the transformants

The aomsas disrupted mutants (aoDmsas) were initially selected on YES medium (20 g/L of yeast extract, 1 M sucrose) supplemented with 150 lg/mL of hygromycin B, while the genetically complemented mutants (ao+msas) were selected on YES medium supplemented with 120 lg/mL phleomycin. These mutants were further screened through PCRs, using two hph gene specific prim-ers hph2R and hph2F (Table 1) and aomsas gene specific primers MSdF1, MSdR1 and MSdR. Total genomic DNA from wild type A. westerdijkiae, aoDmsas and ao+msas strains were digested with enzymes BalI and SmaI and were subjected to southern hybridization [START_REF] Southern | Detection of specific sequences among DNA fragments separated by gel electrophoresis[END_REF] to confirm aomsas gene disruption and genetic complementation. These restriction enzyme were chosen because they cut the 5 0 -flanking region of the aomsas (position 2054 bp and 2850 bp) but do not cut the hph cassette or 3 0 -flanking end of the aomsas gene (Fig. 4b). The digested DNA was then transferred to nylon membrane (Amersham, France) and probed with radioactively labeled DNA fragments of aomsas1 gene ''PM" amplified through primer pair 3 0 MsF/3 0 MsR (probe position is shown in Fig. 4b).

Extraction of secondary metabolites

For secondary metabolites extraction of wild type A. westerdijkiae, aoDmsas and ao+msas mutants, 30 mL filtrate sample of the culture medium was acidified with 200 lL of 12 N HCl, mixed with 30 mL chloroform and vigorously shaken for 10 min. The solvent phase was then decanted, dried under vacuum and re-dissolved in 0.5 mL methanol. Twenty microliters of the sample was then further analyzed by HPLC.

High-performance liquid-chromatography (HPLC) analysis

The HPLC apparatus consisted of a solvent delivery system, with both fluorescence (k ex = 332 nm; k em = 466 nm) and UV detectors (BIO-TEK, Milan, Italy). The analytical column used was a 150 Â 4.6 mm Uptisphere 5 lm C18 ODB fitted with a guard column of 10 Â 4 mm. The column temperature was 30 °C. Kroma 3000 (BIO-TEK) was the data acquisition system. Injections were done with an auto-injector (BIO-TEK, Milan, Italy) and the injection volume was 20 lL. The samples were analyzed by linear gradient elution using 0.2% glacial acetic acid in 99.8% water (v/v) (A) and 100% acetonitrile (HPLC grade) (B). The crude extract was analyzed using a linear elution gradient over 45 min at a flow rate of 1 mL/ min, starting from 10% to 50% solvent B over the first 30 min, continued by a linear gradient to 90% of B in 5 min, followed by an isocratic flow of 90% solvent B for 8 min, and a return to initial conditions over the last 2 min of the run.

Primer name

Sequence (5 Secondary metabolites of A. westerdijkiae were detected by comparing the elution time and maximum absorption of UV with the standard isoasperlactone and asperlactone (Alexis-biochemicals, France) and 6-methylsalicylic acid (ACROS, USA). These standards were used at a concentration of 10 lg/mL. 6-Methylsalicylic acid (maximum absorption k max = 205, 240, 302 nm) were released at 5 min and isoasperlactone/asperlactone (maximum absorption k max = 220 nm) were released between 7 min and 9 min.

0 -3 0 ) LC3 GCIGA(A/G)CA(A/G)ATGGA(T/C)CCICA LC5c GTIGAIGTIGC(G/A)TGIGC(T/C)TC MS1 GCCGCCGGCGTTGACCCGATGAC MS2 CGGCCIAAGGIAA(T/C)ICCITG MS3 CTTAGCAAATCATTCACCATGGAC MS4 (C/T)T(G/C)(A/G)(T/C)(C/T)(A/T)TCCAGCACGCCIGC MS5 ATCCAAACGCTAGACCAACTCGGC MS6 CATTTTCGATCGCCTGCCATGCC MSdF1 ATGCCTTTCTTGGATCCGTCCTCG MSdR1 CTAGGCAGCTAGTTTCTCCGCGAAC MSdF CCGATCGTGTTCAGATCTTGACCTTCCTGATG MSdR ACTAAGTTGGGGATGTCCTGAGATGACGAGA AoLC35-2R CTGGAGGATCTCGCTGATGT AoLC35-2L TTTGATCGACCATTGTGTGC 3 0 MsR CGGGGGCAATGAGATATGAAAAAG 3 0 MsF GAACCCGCTCGTCTGGCTAAG hph2R CTGGATGCAGCAACCTCTAT hph2F CTGAGATGACGAACTTGAC TubF CTCGAGCGTATGAACGTCTAC TubR AAACCCTGGAGGCAGTCGC

Chemical complementation study of aoDmsas mutants

The aoDmsas mutants were inoculated in 250 mL Erlenmeyer flask containing 100 mL SAM medium and incubated at 25 °C. After 72 h 0.7 mM each of 6-MSA, aspyrone and diepoxide were added independently to separate growing cultures of aoDmsas mutants and again incubated at 25 °C without shaking. After 4 days from precursor addition secondary metabolites were extracted for HPLC analyses.

Sequence accession number

The sequence obtained was deposited in GenBank under the Accession No. AY540947.

Results

aomsas is a MSAS-type polyketide gene of A. westerdijkiae

With the intent of extending the 700 bp KS domain fragment of a MSAS-type PKS gene identified by [START_REF] Atoui | Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase gene in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A[END_REF] in A. westerdijkiae, we adopted the degenerated primers based gene walking and RACE-PCR techniques. The specific primers MS1, MS3, MS5 and MS6 and degenerated primers MS2 and MS4 (Table 1) allowed the sequencing of a complete 5298 bp aomsas gene (Fig. 2). The aomsas gene displayed a unique open reading frame (ORF) of 1766 amino acids. Alignment of the amino acid sequence of aomsas with other PKS gene displayed an identity of 56% to 6-MSAS gene involved in the biosynthesis 6-MSA in Byssochlamys nivea, 54% to 6-MSAS gene involved in the biosynthesis of patulin in P. patulum and 50% to a hypothetical 6-MSAS gene in A. terreus (Fig. 2). The aomsas also displayed a significant identity of 40% with the PKS responsible for orsellinic acid biosynthesis in Streptomyces viridochromogenes. Like other 6-MSAS gene, aomsas contained characteristic conserved domains of fungal type I PKSs (Fig. 2). These domains were (from N terminus to C terminus) b-ketoacyl synthase, acyltransferase, dehydratase, b-ketoacyl reductase, and an acyl carrier protein. No thioesterase or enoyl-reductase domains were found in aomsas gene.

Asperlactone and isoasperlactone productions and aomsas gene expression in A. westerdijkiae

In liquid synthetic medium (SAM), without shaking at 25 °C, A. westerdijkiae presented two exponential growth phases from day 3 to day 5 (early exponential growth phase) and day 6 to day 9 (late exponential growth phase), followed by a stationary phase from day 9 onward (Fig. 3a). Asperlactone and isoasperlactone were simultaneously excreted from day 4 and reach a maximum level at day 9 and day 10, respectively. We have further observed that 6-MSA was transitorily produced in very low quantity between day 6 and day 8 and then disappeared (Fig. 3a).

Tracking the transcriptional signals of aomsas gene during different growth stages of A. westerdijkiae in SAM medium, we In the wild type a 2.8 kb band was detected. In transformant ectMS the 2.8 kb band is intacted and an additional band was revealed showing an ectopic insertion of the replacement construct. In the transformants aoDmsas1 and aoDmsas1 the 2.8 kb band is replaced by the expected 5.2 kb band. In the genetically complemented mutant ao+msas the 5.2 kb band is replaced by the wild type 2.8 kb band.

observed that expression of the gene started from day 3 onwards, stayed constant at maximum level between day 4 and 9 and then stopped after day 11 (Fig. 3b).

aomsas gene code for 6-MSA, isoasperlactone and asperlactone in A. westerdijkiae

Following transformation of A. westerdijkiae with TopoMShph vector (Fig. 4b), one hundred and twenty transformants were obtained. Forty transformants were screened by two consecutive PCRs on genomic DNA in order to monitor the integration of hph gene into the genome of A. westerdijkiae. In only two out of the selected forty transformants the hph cassette was detected by the two PCR tests (5% transformation efficiency was obtained). Digested genomic DNA from the wild type A. westerdijkiae and the two aoDmsas transformants were subsequently analyzed by southern blotting. In the wild type A. westerdijkiae a signal corresponding to 2.8 kb fragment of aomsas gene was observed when its digested genomic DNA was probed with PM (Fig. 4c, lane 1). Probing of the aomsas disrupted mutants, i.e. aoDmsas1 and aoDm-sas2, with PM probe resulted into an expected signal of 5.2 kb (2.8 kb correspond to aomsas + 2.4 kb correspond to hph cassette) (Fig. 4c, lane 3 and 4, respectively). We have also observed one ectopic mutant, i.e. ectMS, where the transformation construct was inserted into a non targeted region (Fig. 4c, lane 2).

Wild type A. westerdijkiae, aoDmsas1, aoDmsas2 and ectMS, were grown on solid CYA and liquid SM media, no difference was observed in their colony sizes, growth patterns, visual pigments and conidial counts. Filtrate of the 10 days old culture of wild type A. westerdijkiae, aoDmsas and ectMS mutants were used to extract secondary metabolites. The HPLC profiles of the extracted secondary metabolites revealed that inactivation of aomsas gene by the insertion of hph cassette disrupt the production of isoasperlactone, asperlactone (Fig. 5a) and the transitory biosynthesis of 6-MSA. No impact of the ectopic insertion was observed on the biosynthesis of secondary metabolites (data not shown).

To confirm the role of aomsas gene, genetic complementation of the two aoDmsas mutants were performed with pAN-MSAS transformation vector (containing aomsas gene fragment and phleomycine gene cassette). Filtrate of the 10 days old culture of positively tested complemented mutant ao+msas were used to extract secondary metabolites. HPLC profile of ao+msas mutant revealed that re-insertion of aomsas gene restored the productions of asperlactone, isoasperlactone and 6-MSA (Fig. 5a).

6-MSA, diepoxide and aspyrone are intermediates of isoasperlactone and asperlactone

Chemical complementation was performed by independent incorporation of 6-MSA (0.7 mM), diepoxide (0.7 mM) and aspyrone (0.7 mM) to separate growing cultures of aoDmsas mutants. HPLC profile and UV spectra of the chemically complemented mutants revealed that addition of 6-MSA, diepoxide and aspyrone produced similar effects and restored the biosynthesis of both isoasperlactone and asperlactone (Fig. 5b).

Discussion

From the analyses of fungal PKS gene sequences, [START_REF] Bingle | Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes[END_REF] suggested that these genes could be divided into two subclasses designated as WA-type and MSAS-type or partially reduced type. They designed two pairs of degenerated primers, i.e. LC1/LC2c and LC3/LC5c for the amplification of fungal PKS genes in each of these subclasses. We have previously utilized LC3/LC5c pair of primer to clone a 700 bp KS domain of aomsas gene in A. westerdijkiae [START_REF] Atoui | Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase gene in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A[END_REF]. After prolongation of this fragment by gene walking approach to a complete 5298 bp gene and alignment in database with other PKS genes, we have observed that the amino acid sequence of aomsas has high degree similarities to fungal MSAS-type PKS genes (Fig. 2). The different conserved domains identified in aomsas further confirmed that this gene belongs to the MSAS-type subclass (Fig. 2). These observations are in accordance to the findings of [START_REF] Bingle | Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes[END_REF], who stated that the degenerated pair of primers LC3/LC5c has the capability to amplify fungal MSAS-type PKS genes.

To understand the role of aomsas gene in A. westerdijkiae, we first investigated the kinetic production of secondary metabolites and the expression of the aomsas gene. We have observed that both transcriptional signals of aomsas and biosynthesis of asper- westerdijkiae. HPLC traces of the lactonic metabolites, i.e. isoasperlactone (eluted at lactone, isoasperlactone and 6-MSA occur within the exponential growth phase of A. westerdijkiae (Fig. 3). These observations suggest that aomsas could be related to the biosynthesis of asperlactone and/or isoasperlactone and/or 6-MSA. It has been reported in the literature that MSAS-type PKS genes are involved in the biosynthesis of several olyketide metabolites varying greatly in their functions. An MSAS-type PKS gene 6msas has been found to be involved in the biosynthesis myctoxine patuline in P. patulum, P. urticae and Byssochlamys fulva [START_REF] Beck | The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases[END_REF][START_REF] Bu'lock | Regulation of 6-methylsalicylate and patulin synthesis in Penicillium urticae[END_REF][START_REF] Puel | The inability of Byssochlamys fulva to produce patulin is related to absence of 6-methylsalicylic acid synthase and isoepoxydon dehydrogenase genes[END_REF]. Another MSAS gene AviM has been found to be involved in the biosynthesis of antibiotic avilamycin in S. viridochromogenes [START_REF] Gaisser | Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57[END_REF]. The MSAS gene has also been reported to induce the production plant defense compound salicylic acid in tobacco [START_REF] Nasser | Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco[END_REF]. Although A. westerdijkiae is a non-producer of patuline, avilamycin and salicylic acid but this do not preclude the possibility of having an MSAS-type PKS gene in its genome. A precedent of such a situation is provided by [START_REF] Geisen | Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi[END_REF], who used a multiplex PCR method to identify sequences homologous to aflatoxin biosynthesis gene in the non-producing species of Aspergillus and Penicillium [START_REF] Geisen | Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi[END_REF].

Insertional inactivation remained the key technique for functional characterization of various fungal pks genes [START_REF] Atoui | Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase gene in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A[END_REF][START_REF] Bacha | Aspergillus westerdijkiae polyketide synthase gene ''aoks1" is involved in the biosynthesis of ochratoxin A[END_REF][START_REF] O'callaghan | A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus[END_REF]. So we followed similar technique to produce aomsas knockout mutants ''aoDmsas1" and ''aoDmsas2". These mutants were found deficient in the biosynthesis of two end product lactonic metabolites, i.e. isoasperlactone and asperlactone (Fig. 4a), and one compound of intermediate nature, i.e. 6-MSA. These observations confirmed that aomsas played an important role in the biosynthesis of 6-MSA, isoasperlactone and asperlactone. We further proved these results by performing genetic complementation, where the genetically complemented mutant ao+msas restored the biosynthesis of all the missing metabolites (Fig. 4a).

It has been previously demonstrated through NMR studies that diepoxide and aspyrone are intermediates in the biosynthetic pathway of isoasperlactone and asperlactone in A. melleus (Fig. 1) [START_REF] James | 17O NMR in biosynthetic studies: aspyrone, asperlactone and isoasperlactone, metabolites of Aspergillus melleus[END_REF]. These authors further stated that asperlactone is directly biosynthesized from the diepoxide, while in the biosynthesis isoasperlactone the diepoxide is first converted to aspyrone and then aspyrone to isoasperlactone. They also suggested an alternate rout for the biosynthesis isoasperlactone without the involvement of aspyrone. Chemical complementation of the asperlactone/isoasperlactone deficient mutant, i.e. aoDmsas, through incorporation of 6-MSA, diepoxide and aspyrone clearly shown that, these products are intermediates in the biosynthetic pathway of asperlactone and isoasperlactone (Fig. 5b). Indeed each of the incorporated metabolite restored simultaneously the biosynthesis of both asperlactone and isoasperlactone. Aspyrone and diepoxide are never observed in the culture medium of wild type A. westerdijkiae. This could probably be due to a quick conversion of these compounds to their final products (asperlactone and isoasperlactone), avoiding an intracellular accumulation and extracellular excretion.

Based on our results and the findings of James and Andrew, (1991) we proposed a biosynthetic scheme for the biosynthesis of isoasperlactone and asperlactone (Fig. 1). According to this scheme, during the early biosynthetic steps aomsas gene induces the production of 6-MSA, which is then converted to diepoxide. The diepoxide could then follow two routes, one that leads directly to the production of asperlactone while the second leads to the production of isoasperlactone via aspyrone pathway. We have observed during chemical complementation experiment that even incorporation of aspyrone without diepoxide restored the production asperlactone and isoasperlactone in aoDmsas mutant. Previously [START_REF] James | 17O NMR in biosynthetic studies: aspyrone, asperlactone and isoasperlactone, metabolites of Aspergillus melleus[END_REF] did not proposed this scheme; they proposed aspyrone as intermediate of isoasperlactone but not of asperlactone. Hence we proposed that diepoxide and aspyrone could be enter-convertible compounds which could lead to the production of both asperlactone and isoasperlactone. Our observations are in accordance to the previous findings, suggesting that lactonic metabolites asperlactone and isoasperlactone could be derived from a common biosynthetic precursor, a diepoxy intermediate [START_REF] Sorensen | Toxicity of penicillic acid for rat alveolar macrophages in vitro[END_REF].

This new aomsas gene can be used for further experiments, as a starting material for the identification of asperlactone/isoasperlactone gene cluster.

Fig. 1 .

 1 Fig. 1. Proposed biosynthetic scheme of isoasperlactone and asperlactone in A.westerdijkiae. Based on our results and the study of[START_REF] James | 17O NMR in biosynthetic studies: aspyrone, asperlactone and isoasperlactone, metabolites of Aspergillus melleus[END_REF], we proposed a hypothetical biosynthetic scheme of isoasperlactone and asperlactone biosynthesis. Straight arrow lines indicate our proposed scheme, dotted arrow lines indicate biosynthetic scheme of[START_REF] James | 17O NMR in biosynthetic studies: aspyrone, asperlactone and isoasperlactone, metabolites of Aspergillus melleus[END_REF], while dashed arrow line indicate steps that are shared in our proposed biosynthetic scheme and in the scheme of[START_REF] James | 17O NMR in biosynthetic studies: aspyrone, asperlactone and isoasperlactone, metabolites of Aspergillus melleus[END_REF].

Fig. 2 .

 2 Fig. 2. Alignment of the deduced amino acid sequence of aomsas gene with other MSAS-type PKS gene. 6-MSAS involved in the biosynthesis 6-MSA in Byssochlamys nivea (Accession No.: AAK48943), 6-MSAS involved in the biosynthesis of patulin in P. patulum (Accession No.: CAA39295), 6-MSAS in A. terreus (Accession No.: AAC49814) and PKS responsible for orsellinic acid biosynthesis in Streptomyces viridochromogenes (Accession No.: AAK83194). Red boxes indicate conserved regions of different functional domains. Flash arrows indicate positions of different specific and degenerated primers (Table 1) used in gene walking experiment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .

 3 Fig. 3. Kinetic production of secondary metabolites and expression of aomsas gene in A. westerdijkiae. (a) Kinetic production of isoasperlactone, asperlactone and 6-MSA in a growing culture of A. westerdijkiae at 25 °C in synthetic medium during a time course of 2-18 days. (b) Profile of aomsas gene expression (upper panel) by RT-PCR. Beta tubuline was used as loading control (bottom panel).

Fig. 4 .

 4 Fig. 4. Schematic representation of aomsas gene disruption in A. westerdijkiae. (a) Using primer pair MsdF/MsdR (Table 1), 1995 bp aomsas fragment containing SalI restriction site (indicated by triangle) was amplified from a 5.298 kb aomsas gene. PCR product was cloned into pCR2.1-Topo plasmid to generate plasmid TopoMS. pID2.1 plasmid vector was restricted with SalI (indicated by triangle) to obtain hph cassette (2.4 kb). TopoMS was restricted with SalI and ligated with hph cassette to genarate TopoMShph transformation vector. Different colors on the aomsas gene indicate different functional domains, i.e. b-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), b-ketoacyl reductase (KR), and an acyl carrier protein (ACP). (b) Protoplasts of A. westerdijkiae were prepared and aomsas gene was disrupted using TopoMShph vector to obtain aoDmsas mutants. The small red line below aoDmsas indicates position of radioactively labeled probe, i.e. PM: aomsas gene specific probe amplified by using primer pair AoLC35-2R/AoLC35-2L. Positions of the restriction sites of SmaI and SalI are indicated by triangles. (c) Genomic DNA of wild type A. westerdijkiae, aoDmsas and ao+msas transformants were digested with SmaI and BalI restriction enzymes and probed with radioactively labeled aomsas fragment (PM).In the wild type a 2.8 kb band was detected. In transformant ectMS the 2.8 kb band is intacted and an additional band was revealed showing an ectopic insertion of the replacement construct. In the transformants aoDmsas1 and aoDmsas1 the 2.8 kb band is replaced by the expected 5.2 kb band. In the genetically complemented mutant ao+msas the 5.2 kb band is replaced by the wild type 2.8 kb band.

Fig. 5 .

 5 Fig. 5. Secondary metabolites study. (a) Secondary metabolites were extracted from 10 days old cultures of wild type, aoDmsas and ao+msas strains of A. westerdijkiae. HPLC traces of the lactonic metabolites, i.e. isoasperlactone (eluted at 6 min) and asperlactone (eluted at 9 min) were compared. (b) Separate growing cultures of aoDmsas mutant in synthetic medium at 25 °C were independently complemented with 0.7 mM 6-each of methylsalicylic acid, aspyrone and diepoxide. Secondary metabolites were extracted from 8 days old cultures and analyzed by tracking the HPLC traces.

  4 Á7H 2 O, 10 mL mineral solution (composition per liter of distilled water: 70 mg Na 2 B 4 O 7 Á10H 2 O, 50 mg (NH 4 ) 6 Mo 7 O 24 Á4H 2 O, 1000 mg FeSO 4 Á7H 2 O, 30 mg CuSO 4 Á5H 2 O, 11 mg MnSO 4 ÁH 2 O, 1760 mg ZnSO 4 Á7H 2

Table 1

 1 Oligonucleotide primers.

min) and asperlactone (eluted at 9 min) were compared. (b) Separate growing cultures of aoDmsas mutant in synthetic medium at 25 °C were independently complemented with 0.7 mM 6-each of methylsalicylic acid, aspyrone and diepoxide. Secondary metabolites were extracted from 8 days old cultures and analyzed by tracking the HPLC traces.

Acknowledgment

The authors are grateful to the Higher Education Commission of Pakistan for according PhD scholarship to Mr. Nafees Bacha.