5. Screening for selective media for Xiaman-huakuii rhizobia
Abstract: The data from the diversity study have been used to
screen for selective agents. Selectivity of media confirmed by
various authors are confirmed and the use of triphenylmethane dyes
as selective agents seem to be promising for the Xiaman-huakuii
group. A tentative medium for the fast verification of Xiaman
isolates is proposed.
5.1.1. Screening of potential agents for rhizobia: Overall selectivity was assessed by PROG_GC.PRG using including the data from the soil sample control plates. The soil samples mentioned in sect. (4.1.4.) were from the supernatant of 14 soil samples collected from different habitats in the southern suburbs of Chengdu and from the supernatant of 14 soil samples collected on the Xiaman grasslands (including river plain, northern slope, southern slope etc. as well as a sample from Hongyuan 70 km to the south). The grassland soils had been frozen for 10 months before use, Chengdu soils were freshly collected. After preparation, 15% sterile glycerol was added to each of the supernatant solutions which have been stored at -20oC. When pouring the agar plates, two plates -one with Chengdu soil and one with grassland soil supernatant- were made. Of the data in Table T-4.1-A, the data for the too closely related contaminants (5-20-26-53) was discarded. Afterwards, selectivity for rhizobia (rhizobial selectivity coefficient, RSC) was calculated as:
( Number of rhizobia strains growing on it/number of total
rhizobial strains
- 0.5*(Number of control strains growing on it/number of control
strains)
- Chengdu soil bacteria growing on it/Chengdu soil bact. on control
plate[as ln/24,min:-0.25]
- Grassland soil bact. growing on it/Grassl. soil bact. on control
plate[as ln/24,min:-0.25] )*100
A high RSC (e.g. one above 50) implies selectivity for rhizobia, a low RSC means selectivity against rhizobia.
5.1.2. Screening of potentially selective agents for indivual clusters: From these data, we also paid attention to agents selective for individual clusters.
5.1.3. Repetition of some of the carbon media: Methods identical to section 4.
5.1.4. Combination of selective agents: A mildly selective solution (MILSEL) was combined of (bacitracin 50, penicillin 3, bromothymol blue 20, nalidixic acid 50, CoCl2 10 on YMA). For grassland rhizobia a Xiaman/Sinorhizobia-selective solution (SELSOL) was combined of (MILSOL*1,malachite green 20,SeO2 300 on YMA). Other combined media were z01 (chloramphenicol 10, bromthymol blue 10, CoCl2 10 on lactate), z02 (chloramphenicol 10, bromthymol blue 10, CoCl2 10 on YMA), z03 (penicillin 5, nalidixic acid 50, bromothymol blue 10, bacitracin 50 (this time added after autoclaving), CoCl2 10 on YMA), z04 (penicillin 3, bacitracin 50 (this time added after autoclaving) on YMA), z05 (erythromycin 20, penicillin 3, SeO2 50, bromothymol blue 5,malachite green 10,nalidixic acid 50, CoCl2 10 on YMA) and z06 (penicillin 3, SeO2 50, bromothymol blue 5,malachite green 10,nalidixic acid 50, CoCl2 10 on YMA). Methods as in section 4.
------------------------------------------------------Table T-5.2-A: Selection of potentially selective agents for different clusters
-------------------------------------------------------
RSC substance fast-growing rhizobia vs. non-rhizobia
-9 T03 growth at 4oC 1/20 10/19
-7 C03-asparagine-DL 5/20 13/19
-6 C01-ascorbic acid 0/20 4/19
-3 M18 VO3NH4 2000 6/20 4/19
0 R03 NaCl 1500 0/20 12/19
67 A05 penicillin 5 18/19 12/18
69 C28-sorbose 18/21 10/19
70 C14-lactate-DL Na 18/21 11/19
71 M03 CoCl2.H2O 10 21/21 13/19
75 T02 growth on bromoth b20 18/21 11/19
76 R05 bromothymol blue 750 16/21 10/19
78 NM2 SELSOL*0.05 + YMA 5/7 1/7
79 C08-erythritol-meso 18/21 10/19
104 R09 malachite green 20 16/20 7/19
vetch rhizobia vs. non-rhizobia
68 C18 maltose 6/6 14/19
69 z11 lactate+chlora+bro+Co 3/4 6/14
70 C17-malonate Na 6/6 11/19
71 C30-tartrate-L Na 6/6 11/18
71 M03 CoCl2.H2O 10 6/6 13/19
73 T02 growth on bromoth b20 5/6 11/19
74 R09 malachite green 20 3/6 7/19
78 z04 pen+bactitr 3/3 8/13
83 C28-sorbose 6/6 10/19
84 C14-lactate-DL Na 6/6 11/19
93 C08-erythritol-meso 6/6 10/19
99 R05 bromothymol blue 750 6/6 10/19
Xiaman rhizobia vs. non-rhizobia
67 M29 TiO2 10000 7/7 15/19
67 R02 NO3Na 2000 7/7 15/19
68 R11 nalidixic acid 200 6/7 7/19
68 C18 maltose 7/7 14/19
69 M01 AlCl3.6H2O 300 6/6 13/19
69 C13-inositol 7/7 13/19
70 C14-lactate-DL Na 6/7 11/19
70 C17-malonate Na 7/7 11/19
71 C30-tartrate-L Na 7/7 11/18
71 z08 lactate (pH=8.5) 2/2 3/6
71 M03 CoCl2.H2O 10 7/7 13/19
72 z03 pen+nal+bromot+bacitr 4/7 2/14
72 A05 penicillin 5 7/7 12/18
72 C16-malate-DL 7/7 11/17
75 T02 growth on bromoth b20 6/7 11/19
80 z06 malg+bromot+Se+Co+bac 3/7 1/14
80 C02-anthrone 7/7 12/19
83 C28-sorbose 7/7 10/19
91 NM1 MILSOL*0.05 + YMA 2/2 1/7
106 NM2 SELSOL*0.05 + YMA 2/2 1/7
124 R09 malachite green 20 6/6 7/19
Halophiles/Sinorhizobia vs. non-rhizobia
67 M29 TiO2 10000 5/5 15/19
67 R02 NO3Na 2000 4/4 15/19
68 R07 congo red 3000 5/5 15/18
69 T02 growth on bromoth b20 4/5 11/19
69 M01 AlCl3.6H2O 300 5/5 13/19
71 M03 CoCl2.H2O 10 5/5 13/19
72 A05 penicillin 5 4/4 12/18
79 R05 bromothymol blue 750 4/5 10/19
82 R11 nalidixic acid 200 5/5 7/19
90 M16 SeO2 300 5/5 10/19
93 C08-erythritol-meso 5/5 10/19
94 R12 berberine HCl 275 3/5 6/19
104 R09 malachite green 20 4/5 7/19
115 A03 erythromycin 50 5/5 5/18
Bradyrhizobia vs. non-rhizobia
67 C28-sorbose 5/6 10/19
68 A02 chloramphenicol 20 5/6 12/19
68 C18 maltose 6/6 14/19
69 M01 AlCl3.6H2O 300 6/6 13/19
71 M03 CoCl2.H2O 10 6/6 13/19
73 T02 growth on bromoth b20 5/6 11/19
76 C08-erythritol-meso 5/6 10/19
78 z04 pen+bactitr 4/4 8/13
80 z02 chloram+bromotbl+Co 5/5 8/14
84 C14-lactate-DL Na 6/6 11/19
-------------------------------------------------------
Note: for the detailed data, see table T-4.1-A
-------------------------------------------------------
5.2.1. Selective agents for all rhizobia: Substances showing selectivity for a broad range of rhizobia are bromothymol blue, CoCl2, penicillin and erythritol. Malachite green is a substance very promising for most fast-growing rhizobia and overall scoring very well, it is however too inhibitive too some individual strains.
5.2.2. Selectivity for individual clusters: For the Sinorhizobia, furthermore erythromycin, SeO2 and berberine are of interest. For the Xiaman cluster (Astragalus and Onobrychis), furthermore anthrone, sorbose and use of organic acids might be taken into consideration. However, malachite green is too inhibitive for many vetch and all slow-growing rhizobia. For the vetch rhizobia, C source utilization and antibiotics resistance seem to be the only alternative. The case is also difficult for bradyrhizobia, where Co, Al and chloramphenicol resistance seem to be most promising.
5.2.3. Feasability of C/N source utilization media: Despite some C-source utilization data got quite good scores, they are problematic in that the growth scored after 9 days is often very poor (e.g. melezitose, sorbose, erythritol). Furthermore, it has been difficult to reproduce these results: for example, a repetition of the erythritol medium resulted in a much better growth of the control strains. In some cases, it was the use of carbon sources seemed to be feasible only for a very low proportion of the rhizobia blotted (e.g. especially visible with melezitose (z09)).
5.2.4. Results of combined media: Some of the combined media (such as z05,z06) showed too selective, whilst others had a too low selectivity. Only for the bardyrhizobia did combined media show superior over individual substances, whereas the high scoring of NM1 and NM2 for the Xiaman-huakuii rhizobia probably only reflects the selectivity of malachite green.
5.3.1. Reproducibility of tests reported in the literature: The reproducibility of tests can be a difficult problem for phenetic studies (#On 1991). As well is the influence of metal ion concentration highly dependent on the presence of other (precipitating) ions or chelators (#Hughes 1991), for example it even can make a difference whether a chloride or a sulfate salt is used (#Biro 1995). However it seems that the "standard" YMA medium (employed by #Sadowsky 1983, #Chen 1987, #Zhang 1991, #Sun 1993, #Novikova 1994, #Chen 1995 and many others), is successful in ensuring some comparability at least for resistances: Most screening results simply confirmed the reports of previous authors, e.g. bradyrhizobia indeed have been reported as chloramphenicol (#Gault 1993) and Co2+/Al3+ resistant (#Tong 1994), Sinorhizobia as selenite-(#Kinkle 1994) and erythromycin-resistant (#Chen 1988) and a combination of penicillin and bacitracin had been employed for the isolation of vetch rhizobia has been employed by #Louvrier (1995). The use of erythritol as C source had previously been advocated for a selective medium for Agrobacterium radiobacter biotype II by #New (1971). However, most authors have found carbon sources, even if selective, to have detrimental effects on growth speed (#Louvier 1995).
5.3.2. Dye resistance seems to be promising for Xiaman-huakuii rhizobia verification: The result that Xiaman and many other fast-growing rhizobia are resistant against the triphenylmethane dye malachite green brings this thesis in line with the results of the #Pattison (1974) who also reported malachite (=brilliant) green to be a potent selective agent for all fast-growing rhizobia. A related compound, crystal violet, was the first agent to be advocated by #Churchman (1912) and #Anderson (1929); #Fred (1932) reviews this agent to have been used in concentration varying from 7 to 200ppm. For rhizobia isolated from Nanjing Astragalus sinicus, #Cao (1972) reports some strains to be resistant to as much as 10-1000ppm and confirms that temporary growth on 10ppm crystal violet medium did not impair neither nodulation nor nitrogen fixation ability of the three strains tested for these features. Crystal violet at the concentration we employed showed too inhibitive in our screening, whereas malachite green gave optimal results. This result seems to endorse the finding of #Pattison (1974) who at equal concentrations also found crystal violet to be more inhibitory than malachite green. Against malachite-green-resistant-non-rhizobia nalidixic acid (200pm;13/14 vs.4/7) and AlCl3.6H2O (300 ppm; 13/13 vs. 4/7) might be tried. Furthermore, our data suggest that bromothymol blue also might be a valuable agent for the screening for rhizobia. It is advocated that these studies should be repeated with a number of dyes from different suppliers (others than the Chengdu producers) and a wider range of strains. For example when using fresher malachite green produced 1996 in Tianjin, 4 ppm seemed a better choice than 20ppm.
One of these rhizobia-selective agents (crystal violet) had once been considered as agent for the differentiation of different cell wall structures (Gram-stain) and it might be interesting to compare different patterns in dye resistance to taxonomies dependent on cell-wall components.
5.3.3. Fast verification of Xiaman rhizobia: For the fast verification of Xiaman rhizobia at the moment a combined testing with malachite green resistance (R09 with 4PPM, growth should occur), a combined selective solution (NM02, growth should occur) and incubation at 4oC (T03, no growth should occur) is recommended. Additionally growth on citrate or tartrate (T-4.2-C) can be used to ascertain whether it is a Sinorhizobium and growth on LB should score negative for Xiaman-huakuii rhizobia whilst varying for Sinorhizobia.
5.4. Testing whether different rhizobial groups can be differentiated by triphenylmethane dyes
The previous results proposed one of the triphenylmethane dyes as best selective for Qinghai-Tiber rhizobia. However most dye screening was done when only little was known about rhizobial diversity, for example #Anderson (1929) is very hazy about the strains employed. The following experiment should reveal group differences in dye resistance.
5.4.1. Materials and methods: Subsequently, a wider range of rhizobia was tested from the collection at Marburg University. This was done by a similar design, only that now a wider spectrum of strains were subjected to treatments of triphenylmethane dyes, each on TY (#BERINGER 1974) and YMA (#VINCENT 1970). Strains designated as "Vic", "Phas" and "Lotus" were isolated by Pablo Vinuesa Fleischmann at Tenerifa (Canary Islands) from Vicia faba, Phaseolus vulgaris and Lotus sp. Other strains were from the Marburg University reference strain collection. Also some of the Qinghai/Tibet plateau strains (nos. 2..,U...) aforementioned in section 4 were included. The transfer to the plates was either direct by tooth-pick from agar plates ("F") or using a microwell blotting of liquid cultures diluted to a suspension of 1.5-4 million cells/ml ("B") . Malachite green ("MG") and crystal violet ("CV") were applied in concentrations varying from 0-27 ppm, two replicates were done each.
---------------------------------------------------------------------------
Table T-5.4-A: Triphenylmethane resitances of a broad spectrum of rhizobia
----------------------------------------------------------------------------
S C r
t NNN r r C I CCU DT D
r ZZZLLLLLLLL I A IIS SA S r O I
a PPPOOOOOOOO UUUUU 2 A C TKAAD ML M + V r PPPPP R 33 I
i 222TTTTTTTT 10011UUO +-O r 5 T1/ +2LTTA 11 1VVVVIVVVX HHHHH S 00 I
n 000UUUUUUUU 13755DD/OOO/2227 855CC5M667254PH9IIIICIII62AAAAA 5 11 2W
s 233SSSSSSSS /////001///12205 90IFF65119120F27CCCC1CCC/1SSSSS 7 45 33
media 44778122665 5141334455511111 50INN0A1115302C8556907732141223 1 80 54
R. loti---- Qinghai/Tibet--f etli-tro-leguminosarum--------- z Ag Ps
----------------------------------------------------------------------------
BTY0 NNNNOOOYNYO---O-NYOOOONOYNOO-OOOOON-OOO-YYNONNOOYOOYNOOOYOOO---OY-Y-
BTY0 NNNNOOYNOYY---O-NONOOOOOYOOO-YOOOON-NNO-YYONNNOONONYNOONYOOO---OY-Y-
BTY27CV NNNNYYNNNYY---O-NNNYOYNYYNOO-OOONNN-NNN-YNNNNNNNNNNYYNNNYNNN---OO-Y-
BTY27CV NNNNYYNNOYY---O-ONNYOYNOYNOO-OOONON-NNN-YNNNNNNNNNNYNNNNYYNN---OO-Y-
BTY27MG NNNNYYYYNYY---N-NNNOOONYYOYN-OYNNNN-OYO-YNNNNYNNNNNYNNNNYNNN---OO-Y-
BTY27MG NNNNYYYONYY---N-NNNOOYNYYNYO-OYONNN-NYO-YNNNNNNNNNNYYNNNYNNN---OO-Y-
BYMA0 OOONYYYYYYY---Y-NYYYYYYYYYYY-YYYYYY-YYY-YYYNNYYYYYNYYYYYYYYY---YY-Y-
BYMA0 OONNYYYYYYY---N-YYNYYYYYYYYY-YYYYYN-YYY-YYYYNNNYNYNYNYYNYYYY---YY-Y-
BYMA27CVNNNNYYYNYYY---N-NNNYYYNYYNOY-YYYNNO-NNN-YNNNNNNNNNNYNYONYNNN---ON-Y-
BYMA27CVNNNNYYYNNNY---N-NYOYNNNYYNNN-YNNNNN-NNN-YNNNNNNNNNNYNNONNNNY---YN-Y-
BYMA27MGNNNNYYYNNYY---N-NNNYYONYYNYY-YYYNNN-YYY-YNNYNNNNNNNYYNNNYNNN---OY-Y-
BYMA27MGONNNYYNYNYY---N-NNNYYONYYNYY-YYYNNN-YYY-YNNNNNNNNNNYNNNNYNNN---OY-Y-
FTY0 YYYYPPP-P---NOONYP-----Y-----PYP--ONO--YPYYYOOP-PY-P------YO-P----NP
FTY3CV YONYYYY-O---NOYNNY-----Y-----YOO--YYO--YYYYYYNO-ON-Y------OO-Y----YY
FTY3CV YYYOOOO-O---NNOYNY-----Y-----OOO--ONO--YYYYYOOO-OO-O------OO-O----YY
FTY3MG YYYOPYY-Y---NYYNOP-----Y-----NOO--YYY--YPYYYOOP-PP-Y------OO-P----PP
FTY3MG YNNYYYY-Y---YNYNNN-----O-----YOO--NNY--YYYYYYOY-YY-Y------ON-O----YY
FTY9CV YOOYNYY-Y---NNYNNY-----Y-----OOO--YYO--OYYYYNOY-YN-Y------YY-O----YO
FTY9CV YNNYYYY-Y---NNNYNY-----N-----OOO--ONO--YYYYYNOO-YY-Y------YN-O----YG
FTY9MG YYYYOYY-Y---NNYNNP-----N-----YOO--YNN--PYYYYNNN-OY-Y------NN-O----YY
FTY9MG YYYYYYY-Y---NNYNNY-----N-----OOO--NNO--YYYYYNOY-YY-Y------?O-O----YY
FTY27CV YNYYYYO-Y---NNNONY-----Y-----YYN--YYN--YYNOYYNN-NY-Y------NN-N----OY
FTY27CV YNYYYYY-Y---NNNNNY-----Y-----YON--YYN--YYNONNON-NY-Y------NN-O----YY
FTY27MG YOYYYYY-Y---NNNYNN-----N-----YON--YNN--YYYYNNON-YO-Y------NN-O----YY
FTY27MG YYYYNNN-O---NYYOOY-----Y-----OOY--YNN--NYOOYNNO-NN-N------YN-Y----NY
FYMA0 YYYYYNY-Y---YOYNYO-----Y-----YYO--YNO--YYYYYNNN-YY-Y------OY-O----OO
FYMA3CV YYYYYYY-Y---NNYNNY-----N-----YYN--YNY--YYYYYYNY-YY-Y------YO-O----OY
FYMA3CV NYYYYYY-Y---NNYNOY-----N-----YYO--YNO--YYYOYYOY-YY-Y------YY-O----OY
FYMA3MG NYYYYYY-N---NYYNNY-----O-----YYY--YNY--YYYYYYNY-YY-Y------YY-O----YO
FYMA3MG YYYYYYY-Y---NOYNNY-----Y-----YYO--NNN--YYYNYYOY-YY-Y------YY-O----OO
FYMA9CV YYYYYYY-Y---NOYNNY-----O-----NNO--YNY--YYYYYYON-YY-Y------YN-O----YY
FYMA9CV YYYYYYY-Y---ONONNY-----O-----YYO--YOO--YYYYYYYY-YY-Y------YY-N----OO
FYMA9MG YYYYNYY-Y---NNNNNN-----Y-----YYY--YYY--YYYYYYYY-YY-Y------YY-N----OY
FYMA9MG YYYYYYY-Y---NNYNNY-----Y-----YNN--YNN--YYYYYNYY-YY-Y------YY-O----YY
FYMA27CVYYNYYYY-Y---NNNNNN-----N-----YYN--YON--NYNNNNON-YN-Y------ON-N----NN
FYMA27CVYYNYYYY-Y---NNNNNY-----N-----NYY--NNN--NYONNNNO-YY-Y------NN-O----NY
FYMA27MGOOOOYYY-Y---NNNNNN-----O-----OYY--YOY--OOOOONYO-YY-Y------YY-O----OY
FYMA27MGYYYYYYY-Y---NNYNNY-----N-----YYY--NNN--YYYYYNYY-YY-Y------YN-N----PP
----------------------------------------------------------------------------
Notes: "r" replicate,"+" replicate with
five-fold cell concentration, "-" replicate with one-fifth cell
conc. "Y" is pronounced growth, "O" is slight growth, "P" is rugged
colony, "N" is no growth. "tro" is "Rhizobium tropici", "F" fredii,
"z" Azorhizobium caulinodans, "Ag" Agrobacterium (rhizogenes and
tumefaciens), "Ps" Pseudomonas (putida and flavescens)
----------------------------------------------------------------------------
Method of transferring bacteria to plates: The tooth-pick method resulted in higher inocula, so that survival rates were higher, and nearly all colonies survived. A very pronounced influence of inoculum density can also be seen in the data for strains used at different densities, e.g.0-5 in table T-5.4-A.
Choice of medium and dye: the TY medium more often resulted in only slight growth than the sugar-rich YMA medium, but the choice of medium apparently didn't influence survival in both dyes (the death ratio in table T-5.4-B is similar). On dye-containing media growth was more slimy. Both dyes appeared equally inhibitory, no decoloration (#Roth 1992) took place. If only the blotting data are analyzed, on TY MG is slightly more inhibitory and on YMA on CV is slightly more inhibitory than its counterpart, but these data are not significant.
----------------------------------------------- Table T-5.4-B: Growth vigor on different media ------------------------------------------------ medium loti Qingh/T tropic. legumin.all Rhizobiac. BTY0 5-7-2 4-17-3 1-9-0 8-19-1 20-56-8 FTY0 4-4-0 2-3-0 0-3-0 6-7-0 12-20-0 BYMA0 14-0-0 19-0-1 10-0-0 27-0-1 78-0-2 FYMA0 7-0-1 3-2-0 2-1-0 9-1-3 22-6-4 FTY3CV 8-7-1 5-2-3 1-5-0 12-12-2 28-30-2 FTY3MG 12-2-2 4-3-3 1-4-1 15-10-1 35-21-8 FYMA3CV 15-0-1 4-1-5 4-1-0 22-3-1 48-8-8 FYMA3MG 14-0-2 6-2-2 5-1-0 23-1-2 50-6-8 FTY9CV 11-2-3 4-6-0 0-6-0 17-4-3 35-23-6 FTY9MG 15-1-0 3-1-6 1-5-0 17-4-6 37-14-14 FYMA9CV 16-0-0 3-4-3 2-2-2 23-1-2 47-9-8 FYMA9MG 15-0-1 4-0-6 4-0-2 25-0-1 51-1-12 BTY27CV 8-1-5 7-11-6 0-7-3 7-0-21 22-23-35 BTY27MG 11-1-2 7-9-8 2-3-5 6-2-20 28-16-40 FTY27CV 13-1-2 4-0-6 3-1-2 10-3-13 32-6-26 FTY27MG 11-2-3 4-1-5 2-3-1 9-5-12 29-12-23 BYMA27CV10-0-4 10-1-9 4-0-6 7-2-19 32-4-44 BYMA27MG10-0-4 12-2-6 6-0-4 8-0-20 43-3-34 FYMA27CV14-0-2 1-0-9 4-0-2 7-4-15 27-5-32 FYMA27MG12-4-0 2-1-7 5-1-0 19-6-3 40-13-13 ------------------------------------------------Note: Given are the number of strains growing (including replications) for marked (slimy) growth, slight growth and no growth. Only those strains which grow in most control media (YMA0,TY0) are included.
------------------------------------------------
The only significant result is that blotted R.leguminosarum is more inhibited by both dyes than all other rhizobia (P<0.003 for MG, P<0.001 for CV).
5.4.3. Discussion: dye resistance alone is very much influenced by the number of cells applied and thus no ideal single diagnostic criterium. However it is interesting that mesorhizobia are more resistant than the leguminosarum type.