Decreased incidence of disease caused by Sclerotinia sclerotiorum and improved plant vigor of oilseed rape with Bacillus subtilis Tu-100.

Abstract

Sclerotinia sclerotiorum causes serious yield losses in oilseed crops worldwide. Bacillus subtilis Tu-100 significantly reduced (P< or =0.05) the incidence of disease caused by S. sclerotiorum on oilseed rape at harvest in two trials conducted in fields artificially infested with this pathogen. Mean plant dry weight was significantly greater (P< or =0.05) and mean plant length was significantly greater (P< or =0.07) at the seven-true-leaf stage with the Tu-100 treatment than with the control. Mean seed yield per 120 plants at harvest was significantly greater (P< or =0.05) in the second field trial with treatments containing isolate Tu-100. B. subtilis Tu-100 also promoted the growth of hydroponically grown oilseed rape. Plants were approximately 15% greater in dry weight (P< or =0.0001) and 6% greater in length (P< or =0.0025) when grown in the presence of isolate Tu-100 in Hoagland's solution, compared with the noninoculated control. In gnotobiotic studies, the lacZ-tagged strain B. subtilis Tu-100(pUC18) was detected within all roots of oilseed rape. Isolate Tu-100 did not persist in the ectorhizosphere of oilseed rape. Populations of this isolate decreased from 8.5x10(8) colony-forming units (CFU) per seed to approximately 10(2) CFU in the plant ectorhizosphere within 30 days of sowing in autoclaved soil.

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Appl Microbiol Biotechnol (2005) 68: 802–807
DOI 10.1007/s00253-005-1938-x
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Xiaojia Hu.Daniel P. Roberts.Mulan Jiang.
Yinbo Zhang
Decreased incidence of disease caused by Sclerotinia
sclerotiorum and improved plant vigor of oilseed rape with
Bacillus subtilis Tu-100
Received: 23 November 2004 / Revised: 27 January 2005 / Accepted: 8 February 2005 / Published online: 3 March 2005
# Springer-Verlag 2005
Abstract Sclerotinia sclerotiorum causes serious yield
losses in oilseed crops worldwide. Bacillus subtilis Tu-100
significantly reduced (P≤0.05) the incidence of disease
caused by S. sclerotiorum on oilseed rape at harvest in two
trials conducted in fields artificially infested with this
pathogen. Mean plant dry weight was significantly greater
(P≤0.05) and mean plant length was significantly greater
(P≤0.07) at the seven-true-leaf stage with the Tu-100
treatment than with the control. Mean seed yield per 120
plants at harvest was significantly greater (P≤0.05) in the
second field trial withtreatments containing isolate Tu-100.
B. subtilis Tu-100 also promoted the growth of hydropon-
ically grown oilseed rape. Plants were approximately 15%
greater in dry weight (P≤0.0001) and 6% greater in length
(P≤0.0025) when grown in the presence of isolate Tu-100
in Hoagland’s solution, compared with the noninoculated
control. In gnotobiotic studies, the lacZ-tagged strain B.
subtilis Tu-100(pUC18) was detected within all roots of
oilseed rape. Isolate Tu-100 did not persist in the ectorhizo-
sphere of oilseed rape. Populations of this isolate decreased
from 8.5×108colony-forming units (CFU) per seed to ap-
proximately 102CFU in the plant ectorhizosphere within
30 days of sowing in autoclaved soil.
Introduction
Sclerotinia sclerotiorum (Lib.) de Bary is distributed
worldwide and causes disease on oilseed rape and many
other important crops (Boland and Hall 1994; Purdy 1979).
Serious yield losses can result with annual losses to S.
sclerotiorumonoilseedrape(BrassicanapusL.),sunflower
(Helianthus annuus L.), and soybean [Glycine max (L.)
Merr.] estimated at more than U.S. $ 60×106(Lu 2003).
This pathogen overwinters as sclerotia in soil and generally
infects plants as mycelia originating from eruptive ger-
mination of sclerotia in soil or as airborne ascospores that
directly penetrate host tissue (Lu 2003). Diseases caused
by this pathogen are typically initiated on above-ground
plant parts (Abawi and Grogan 1979). On oilseed rape,
stem lesions develop from the soil line or axils of branches
or leaves. Lesions can eventually expand to girdle the stem
and kill the plant (Nyvall 1979).
Oilseed rape is the major oilseed crop in the People’s
Republic of China, with approximately 70×106ha in pro-
duction (Zhao and Meng 2003). Crop rotation to nonsus-
ceptible hosts and fungicide application are the prominent
methods for control of diseases caused by S. sclerotiorum
(Lu 2003; Yu and Zhou 1994). Although several fungicides
areavailable,thesechemicalscanbeexpensive,ineffective,
andhazardous(Lu2003).Therearealsoconcernsregarding
fungicide resistance in populations of S. sclerotiorum
(Gossen et al. 2001).
Traditional oilseed rape breeding programs for disease
resistance have been hampered by a limited gene pool (Lu
2003). Furthermore, selection of oilseed rape cultivars with
low seed glucosinolate content has become a priority for
plant breeders. Glucosinolates are secondary metabolites
present in crops of the Brassicaceae family, some of which
produce toxic compounds when degraded (Zhao and Meng
2003). It is suspected that low glucosinolate levels in seeds
lead to reduced resistance to S.sclerotiorum andother path-
ogens (Liu 2000). Certain degradation products of gluco-
sinolates, especially hydrolysates of indoyl glucosinolate,
have been reported to defend plants from insects and fungal
pathogens on leaves (Brown and Morra 1997; Mithen
X. Hu.M. Jiang.Y. Zhang
Key Laboratory for Genetic Improvement of Oil Crops,
Oil Crops Research Institute,
Chinese Academy of Agricultural Sciences,
Wuhan, 430062, People’s Republic of China
D. P. Roberts (*)
Sustainable Agricultural Systems Laboratory, Henry A. Wallace
Beltsville Agricultural Research Center, United States
Department of Agriculture–Agricultural Research Service,
Beltsville, MD 20705, USA
e-mail: robertsd@ba.ars.usda.gov
Tel.: +1-301-5045680
Fax: +1-301-5046491

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1992).Becauseofthesehealthandenvironmentalconcerns,
and the current breeding trends, alternative control mea-
sures are needed for disease caused by S. sclerotiorum on
oilseed rape in the People’s Republic of China.
Biological control of diseases caused by S. sclerotiorum
receives considerable attention. Coniothyrium minitans
(Campbell) and several other biological control agents sup-
press various diseases caused by S. sclerotiorum on a num-
ber of crops (Adams and Fravel 1990; Escande et al. 2002;
Huang et al. 1993; Li et al. 2003; McQuilken et al. 1997).
Commercial products based on C. minitans, developed for
the control of S. sclerotiorum on oilseed rape and other
crops, are marketed in Europe (de Vrije et al. 2001). These
products are not used in the People’s Republic of China,
possibly due to high cost at current exchange rates. Con-
sequently, there is a need for biological control agents for
the suppression of S. sclerotiorum on oilseed rape that can
be marketed for use in China. We report here decreased
incidence of disease caused by S. sclerotiorum and im-
proved plant vigor of oilseed rape in field trials in China
with treatments containing Bacillus subtilis (Ehrenberg)
isolate Tu-100.
Materials and methods
Bacterial and fungal isolates
B. subtilis Tu-100 was isolated from an oilseed rape rhi-
zosphere (Hu et al. 1992) and shown to inhibit a number
of bacterial and fungal plant pathogens on agar media (Hu,
unpublished data). Isolate Tu-100 was cultured in Luria–
Bertani (LB) broth or agar with or without 30 μg ml−1kana-
mycin. B. subtilis Tu-100 is naturally resistant to 30 μg ml−1
kanamycin. Strain Tu-100(pUC18) was constructed by intro-
ductionofplasmidpUC18(PromegaCorp.,Wuhan,People’s
Republic of China), containing lacZ, into isolate Tu-100 by
electroporation (Chassy et al. 1988). Strain Tu-100(pUC18)
was similar to strain Tu-100 in growth rate, colony mor-
phology, in vitro inhibition of S. sclerotiorum Ss-1, and in-
hibition of isolate Ss-1 in detached-leaf assays (data not
shown). Strain Tu-100(pUC18) was cultured on LB broth or
agar plus 50 μg ml−1ampicillin. Bacterial broth cultures were
incubated at 150 rpm and 28°C. S. sclerotiorum Ss-1 was
isolatedfromasclerotiumformedonoilseedrape.IsolateSs-1
was maintained on potato dextrose agar (PDA) at 4°C. All
microorganisms were obtained from the culture collection of
the Plant Protection Laboratory, Oil Crops Research Institute
(ChineseAcademyofAgriculturalSciences,Wuhan,People’s
Republic of China).
In vitro inhibition assays
For in vitro inhibition assays, one disk (9 mm diam.) of
isolate Ss-1 (from a PDA plate) was added to the center
and four disks (9 mm diam.) of Tu-100 (from a PDA plate)
were added to the periphery of each fresh PDA plate.
These PDA plates were incubated at 22°C for 5 days prior
to measuring the zone of inhibition of hyphal growth. For
this, the distance between the bacterial colony and the
edge of the Ss-1 mycelium was determined. The exper-
iment was performed three times with five replicate PDA
plates.
Detached-leaf assays
Leaves from healthy, greenhouse-grown oilseed rape (B.
napus cv Zhong You 119) plants were detached, washed
with sterile distilled water (SDW), blotted with sterile filter
paper to remove excess water, and air-dried. A single leaf
was detached from each plant. Leaf age, growing position,
and leaf size were similar for all treatments in these ex-
periments. Leaves were spotted with a 0.1-ml suspension
of isolate Tu-100 in SDW [8.6×109colony-forming units
(CFU)ml−1]orwith0.1mlofSDWandincubatedfor1min.
A single, 9-mm-diam. disk of Ss-1 on PDA was subse-
quently applied to detached leaves. Treated leaves were
placed on glass rods that were positioned on moist gauze,
covered with plastic wrap, and incubated at 23°C in a
growth chamber. Area of hyphal growth on each detached
leaf was recorded after 4 days of incubation. For this, the
lengthsofthelongaxisandshortaxiswereaveragedandthe
radius was determined and used in the formula: Area=
π(radius)2. The experiment was performed two times with
seven replicates per treatment.
Plant growth promotion assays
Oilseedrapeseedsweresurface-sterilizedwith0.1%HgCl2,
rinsed thoroughly with SDW, and germinated. Seedlings
with 3- to 5-cm tap roots were secured with sterile cotton in
holes (1.2cm diam.) in covers of sterile containers (18.5 cm
deep, 13.5 cm diam.). A suspension (0.5 ml ) containing
5×109CFU ml−1isolate Tu-100 in LB broth, or 0.5 ml of
sterile LB broth for the control, was added to 1,700 ml of
sterile Hoagland’s solution (Hoagland and Arnon 1938) in
thecontainers.Containerswereincubatedinthegreenhouse
at approximately 16°C (day) and 10°C (night). An addi-
tional 300 ml of sterile Hoagland’s solution was added as
needed over the course of the 90-day experiment. Contain-
ers were tested periodically for contamination by plating on
PDA.Meandryweightperplantandmeanplantlengthwere
determined and compared using SAS Least squares means
(SAS, Cary, N.C.). The experiment was performed three
times with three replicates per treatment. Data from exper-
iments were combined prior to analysis.
Root colonization assays
Experiments to determine the colonization of internal root
tissue by B. subtilis Tu-100(pUC18) were performed es-
sentially as described by O’Callaghan et al. (2000). Seeds
were surface-sterilized with 0.1% HgCl2, rinsed thor-
oughly with SDW, and blotted dry with sterile filter paper.
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Seed (1 g) was incubated with 0.2 ml of a 24-h culture of
Tu-100 (pUC18) in LB broth (9×1010CFU ml−1), or
sterile LB broth for the control, for 20 min at 25°C. Treated
seedsweresownin1%agarplussterileHoagland’ssolution
insteriletubes(18cmlong,1.8cmdiam.)andincubatedfor
21 days at 25°C (day) and 20°C (night) in the growth
chamber. At sampling time, shoots were excised and roots
separated from the agar. Root systems were fixed in glu-
taraldehydeandincubatedinasolutioncontaining5-bromo-
4-chloro-3-indoyl-B-D-galactopyranoside(X-gal)asdescribed
by Boivin et al. (1990). Regions of roots with dark blue
precipitate, due to the degradation of X-gal, were processed
for light microscopy as described by Davey et al. (1993).
Plant cell walls were stained with a phenolic-magenta stain
(Davey et al. 1993). The experiment was performed twice
with ten replicates per experiment.
For ectorhizosphere colonization experiments, oilseed
rape seeds were surface-sterilized and treated with isolate
Tu-100, or sterile LB broth for the control, as above.
Individual oilseed rape seeds, treated with Tu-100 or ster-
ile LB broth, were placed on top of 700 g of an autoclaved
silty clay loam soil (60.4 mg kg−1P, 22.8 mg kg−1N,
147.9 mg kg−1K, 1.38% organic matter) in sterile glass
bottles (18 cm deep, 7 cm diam.), covered with a 0.5-cm
layer of autoclaved soil, and the bottle openings covered
withsterilepaper.Soilwasautoclavedfor1hat121°Cprior
to use. Bottles were incubated at 25°C (day) and 20°C
(night) in the growth chamber. When seedlings emerged,
the paper cover was slit and the bottles were moved to
the greenhouse and incubated at approximately 25°C (day)
and 20°C (night). Plants were watered with sterile water
as needed, through the slit in the bottle cover. At sampling
time,thebottleswerebroken,shootswereexcisedatthesoil
line, and root systems were removed from the soil and
aseptically cut into 2-cm sections. CFU of isolate Tu-100
per root segment was determined by dilution-plating in-
dividual root segments onto LB agar plus 30 μg ml−1
kanamycin. The experiment was performed twice with three
replicates per treatment. Data from experiments were com-
bined for analysis.
Field trials
Field experiments were performed twice over consecutive
years at the Oil Crops Research Institute. The January prior
to seeding, the field (soil characteristics as in root col-
onization assays) was artificially infested with sclerotia of
S. sclerotiorum Ss-1 by imbedding sclerotia in the field at a
depth of 2–3 cm and a rate of ten sclerotia per replicate
block. Three replicate blocks per treatment were arranged
in a random complete block design surrounded by a 1-m
protective belt of oilseed rape plants. Each replicate block
was 3.3×2.0 m with ten rows planted at a density of 10
seeds m−1and rows spaced 33 cm apart. Treatments used
were oilseed rape seed incubated with overnight cultures of
isolate Tu-100 in LB broth or oilseed rape seed incubated
with sterile LB broth. Seeds treated with isolate Tu-100
contained approximately 8.1×108CFU of isolate Tu-100
per seed. Treatments were prepared as in the ectorhizo-
spherecolonizationexperiment,excepttheoilseedrapeseed
was not surface-sterilized. Field trials were conducted from
October through May of each year.
When plants had seven true leaves, five plants were
collected at random from each replicate block and the
plant length and dry weight were determined. After this
sampling, rows were thinned to 12 plants per row. Five
days prior to harvest, 120 plants from each replicate block
were rated for disease incidence. An oilseed rape plant was
considered diseased if one-third of the branches on the
plant contained one or more lesions resulting from infec-
tion by S. sclerotiorum or if the plant contained a lesion on
the caulis (Zhou 1994). At harvest, 120 plants from each
replicate block were sampled for seed fresh weight. Mean
plant length, mean dry weight, mean disease incidence,
and mean seed weight per 120 plants were determined and
compared using SAS Least squares means. Experiments
were analyzed independently.
Results
Inhibition of S. sclerotiorum in vitro and in detached
leaves
Cultures of B. subtilis Tu-100 grown on PDA produced a
metabolite(s) that was released into the agar and inhibited
the hyphal growth of S. sclerotiorum Ss-1. The zones of
inhibition in three separate experiments were 0.92±0.12,
0.90±0.14, and 0.96±0.14 cm. Detached leaves from oil-
seed rape spotted with a suspension of isolate Tu-100 also
had substantially less S. sclerotiorum Ss-1 hyphal growth
than leaves spotted with SDW. Areas of S. sclerotiorum
Ss-1 hyphal growth on leaves spotted with Tu-100 and
SDW were 2.86±0.49 cm2and 8.03±0.61 cm2, respective-
ly,inoneexperimentand3.04±0.23cm2and8.53±0.85cm2,
respectively, in a second experiment.
Plant growth promotion
B. subtilis Tu-100 promoted growth by oilseed rape plants
when grown hydroponically for 90 days in Hoagland’s
solution. Mean dry weight of oilseed rape plants grown in
the presence and absence of isolate Tu-100 was 2.10 g and
1.82 g, respectively, after 90 days. Plants grown in the
presence of Tu-100 were approximately 15% greater in
dry weight than plants grown without this bacterium (P≤
0.0001). Plants grown in the presence of isolate Tu-100
were also slightly greater in length (5%) than plants grown
in the absence of this bacterium (P≤0.0025) after 90 days.
Mean plant length was 23.74 cm and 22.46 cm, respec-
tively, for oilseed rape plants grown in the presence and
absence of isolate Tu-100.
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Root colonization
The lacZ-tagged strain B. subtilis Tu-100(pUC18) was
detected within roots of oilseed rape in gnotobiotic stud-
ies in all ten replicate plants in both experiments. The lo-
cationofstrainTu-100(pUC18)wasevident,duetothedark
pigment formed from the degradation of X-gal by the
activity of β-galactosidase encoded by lacZ on pUC18
(Fig. 1a). Sections of root regions under higher magnifica-
tion confirmed that Tu-100(pUC18) was detected within
root tissue (Fig. 1b). There was no similar dark pigment
present in root sections from non-inoculated controls (data
not shown). Colonization of internal regions was always
in the region of lateral root formation. B. subtilis Tu-100
did not effectively colonize the ectorhizosphere of oilseed
rape plants in experiments conducted in autoclaved soil
(Table 1). Populations of this strain decreased rapidly over
30 days, from 8.5×108CFU per seed to 102CFU in the
entire ectorhizosphere.
Suppression of S. sclerotiorum in the field
B. subtilis Tu-100 effectively suppressed S. sclerotiorum in
field trials conducted over two consecutive years at the
same location with soil artificially infested with this path-
ogen (Table 2). Disease incidence at harvest was signifi-
cantly lower (P≤0.05) in plants treated with isolate Tu-100
than with the control. Plants were harvested approximately
7.5 months after sowing. In addition, plant vigor (deter-
mined 85 days after sowing) was improved in treatments
containing isolate Tu-100. Mean dry weight per plant was
27% and 11% greater with the Tu-100 treatment in ex-
periments 1 and 2, respectively, than with the control treat-
ment. Mean plant length was slightly greater (significant
at P≤0.07) in both experiments. Finally, mean seed yield
per 120 plants at harvest was increased 6% in experiment
1 and 18% in experiment 2 with treatments containing
Tu-100, as compared with the control. There was a signif-
icant increase in mean seed yield per 120 plants (P≤0.05)
in the second experiment in the treatment containing iso-
late Tu-100, relative to the control (Table 2).
Fig. 1 Lightmicrographsshowingcolonizationofinternalregionsof
oilseed rape roots by B. subtilis Tu-100(pUC18). The location of B.
subtilis Tu-100(pUC18) was determined after staining root tissue
with X-gal. Degradation of X-gal by β-galactosidase, encoded by
lacZ on pUC18, resulted in the production of a dark pigment. Root
cells were stained with a phenolic-magenta stain. The size bar
represents 500 μm in a and 35 μm in b. LR Lateral root, Tu-100B.
subtilis Tu-100(pUC18)
Table 1 Distribution of populations of B. subtilis Tu-100 in the
ectorhizosphere of oilseed rape. B. subtilis Tu-100 was applied as a
seed treatment and seeds were sown in autoclaved soil. Seeds treated
with isolate Tu-100 contained 8.5×108CFU of isolate Tu-100 per
seed. ND No CFU of isolate Tu-100 detected, No root root tissue
absent at this distance from the soil line
Root segmenta(cm) Days after plantingb
369 1215 1830
0–2
2–4
4–6
6–8
8–12
4.5±0×105
8.1±0.2×105
No root
No root
No root
3.9±0.3×104
3.9±1.2×104
5.2±0.5×102
No root
No root
5.7±0.7×103
3.5±0.2×102
1.6±0.2×102
3.9±0.6×101
ND
2.4±0.4×103
1.8±0.8×102
3.5±0.9×101
ND
ND
4.9±1.3×102
2.6±0.8×101
ND
ND
ND
3.2±0.4×102
ND
ND
ND
ND
2.3±0.4×102
ND
ND
ND
ND
aDistance from point of excision from the plant. Roots were excised at the soil line
bColony-forming units of isolate Tu-100 were determined by dilution-plating root segments
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Discussion
B. subtilis Tu-100 has beneficial traits that show promise
for use in the production of oilseed rape in China. Isolate
Tu-100 significantly reduced the incidence of disease in
field trials conducted over two consecutive years in fields
artificially infested with S. sclerotiorum. Oilseed rape
plants treated with isolate Tu-100 were also significantly
improved in plant vigor in both trials; and seed yield was
significantly greater in the second field trial.
The mechanisms by which B. subtilis Tu-100 suppressed
the incidence of disease caused by S. sclerotiorum on oil-
seed rape and improved the plant vigor are unknown. How-
ever, cultures of isolate Tu-100 grown on PDA produced a
metabolite(s) that was released into the medium and in-
hibited hyphal growth by this pathogen. Other isolates of B.
subtilis are reported to inhibit bacterial and fungal path-
ogens in plant and animal systems (Logan 1988; Phae and
Shoda 1990; Walker et al. 1998). This inhibitory activity is
attributed in part to the production of antibiotic com-
pounds, including peptides (Banerjee and Hansen 1988;
Paik et al. 1998), lipopeptides (Arima et al. 1968), phenyl-
propanol derivatives (Pinchuk et al. 2002), and a novel
phospholipid compound (Tamechiro et al. 2002). It is also
possible that plant growth promotion resulted in the im-
proved plant vigor observed in the field trials. B. subtilis
Tu-100 promoted the growth of oilseed rape when grown
hydroponically in Hoagland’s solution in the absence of
pathogens.
Strains of Bacillus are among the bacteria commonly
found to colonize internal plant parts (Lilley et al. 1996;
Mahaffee and Kloepper 1997). As with other isolates of
Bacillus, B. subtilis Tu-100 colonized roots of oilseed rape
endophytically (Fig. 1). This bacterium was detected in the
internal portions of roots of every oilseed rape plant sam-
pled in tissues associated with lateral root formation. The
main entry for bacterial endophytes appears to be through
wounds resulting from plant growth, such as in areas of
lateral root formation (Huang 1986; Hallman et al. 1997).
In contrast, isolate Tu-100 did not effectively colonize the
ectorhizosphere of oilseed rape plants (Table 1). Poor col-
onization of the ectorhizosphere occurred in autoclaved
soil, where competition with the indigenous microflora was
reduced. Bacterial endophytes have been shown to be an-
tagonists of plant pathogens in vitro and to suppress plant
disease. However, this report concerning B. subtilis Tu-100
is one of the few where disease suppression by a bacterial
endophyte is confirmed with field trials (Wei et al. 1996;
Hallman et al. 1997).
Future work with B. subtilis Tu-100 will focus on ex-
periments needed to demonstrate the commercial viability
of this bacterium for the suppression of disease caused by
S. sclerotiorum on oilseed rape. Large-scale field trials will
be conducted in China under varying environmental con-
ditions, using a number of cultivars of this crop and a
genetically diverse collection of S. sclerotiorum isolates.
Genetic and biochemical studies will also be conducted to
determine the mechanisms by which isolate Tu-100 sup-
presses disease caused by S. sclerotiorum and promotes
growth by oilseed rape.
Acknowledgement
Natural Science Foundation of China (Project number 39870043).
This project was supported by the National
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Table 2 Biological control of S. sclerotiorum on oilseed rape with
B. subtilis Tu-100 in field trials conducted over two consecutive
years at the Oil Crops Research Institute. Numbers in columns fol-
lowed by the same letter are not significantly different (P>0.05) in
that particular experiment (SAS Least squares means). Seeds were
treated either with overnight cultures of isolate Tu-100 grown in LB
broth or with sterile LB broth. Seeds treated with isolate Tu-100
contained approximately 8.1×108CFU of isolate Tu-100 per seed.
Mean plant dry weight and mean plant length were determined
approximately 85 days after planting. Five plants per replicate block
were randomly sampled and used for these determinations. Mean
disease incidence was determined 5 days prior to harvest. Mean seed
yield per 120 plants was determined at harvest
Experiment Treatment Mean plant dry weight (g) Mean plant length (cm) Mean disease incidence Mean seed yield per120 plants (kg)
1Tu-100
Control
Tu-100
Control
1.61a
1.26b
2.22a
2.00b
19.0a
16.9a
28.0a
25.2a
9.3a
25.7b
6.7a
19.0b
1.60a
1.51a
1.91a
1.61b
2
806