2. 159WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167
of environmental problems, such as water eutrophication
(Chang and Yang 2009; Kang et al. 2011). Enhancement
of plant growth through fertilization to meet the increasing
demands for food has resulted in intense mining of P-con-
taining minerals around the world. It has been estimated
that these P mines could be depleted by 2060 (Gilvert 2009).
Many agricultural soils represent a source of P that is not
readily available to plants but may still be recovered. It has
been suggested that the amount of P in agricultural soils
is sufficient to sustain maximum crop yields worldwide for
about 100 years (Walpola and Yoon 2012).
To use the P accumulated in soils, P-solubilizing micro-
organisms (PSMs) that are able to transform insoluble P to
soluble forms can function as biofertilizers to increase the
soluble P content (Narsian and Patel 2000; Delvasto 2006;
Khan et al. 2007; Zhu et al. 2012). The use of P-biofertiliz-
ers is a promising approach to improve world food security
through enhancing agricultural yield in developing countries
in Africa and Asia, which together account for 50 and 74%,
respectively, of the global land mass and population (Ogbo
2010). Many studies have shown that growth and P uptake
by plants can be enhanced by inoculation of phosphate-sol-
ubilizing fungi (PSF). This effect has been shown in pot
experiments (Vassilev et al. 2006; Mittal et al. 2008) and
under field conditions (Duponnois et al. 2005; Valverde et al.
2006). Among these fungi, P-solubilizingAspergillus species
have been widely studied because of their strong ability to
provide available P and improve plant growth (EI-Azouni
2008; Mittal et al. 2008; Ogbo 2010; Jain et al. 2012; Xiao
et al. 2013). El-Azouni et al. (2008) reported that Aspergillus
niger was able to solubilize and release inorganic P; the
studied strain released 490 μg P mL–1
after 7 d of growth
in Pikovskaya (PVK) medium supplemented with tricalcium
phosphate (TCP). In a pot experiment, inoculation with
A. niger significantly increased plant height by up to 27.5%
and plant dry weight by up to 22.7%, compared with plants
grown in non-inoculated TCP soil. Mittal et al. (2008) iso-
lated six P-solubilizing fungi, two strains of A. awamori and
four strains of Penicillium citrinum, from the rhizosphere of
various crops. When these strains were inoculated onto
chickpea plants in a pot experiment, the two A. awamori
strains had the strongest growth-promoting effects. The
plants inoculated with A. awamori showed a 7–12% increase
in shoot height, a nearly three-fold increase in seed number,
and a two-fold increase in seed weight, as compared with
uninoculated control plants. Ogbo (2010) and Xiao et al.
(2013) reported that biofertilizer produced by A. niger sig-
nificantly (P<0.05) improved the growth of pigeon pea and
wheat plants under culture conditions. Jain et al. (2012)
demonstrated that A. awamori S29 significantly increased
mungbean growth, total P content, and plant biomass in a
pot experiment.
Biofertilizers are usually prepared as carrier-based
inoculants containing effective microorganisms (Accinelli
et al. 2009). However, the local development of commercial
biofertilizers is often restricted by technological limitations
or the scarcity of local sources of peat, the most commonly
used biofertilizer carrier in many countries (Khavazi et al.
2007). The development of locally produced inoculants is
desirable, as they are adapted to local conditions. To do so,
it is important to use carriers and preparation methods that
are widely available and accessible locally. Asuitable biofer-
tilizer carrier should meet the following criteria: (1) it should
be available in powder or granule forms; (2) it should be
able to support microorganism growth and survival, and
easily release functional microorganisms into the soil; (3) it
should have a strong moisture absorption capability, good
aeration characteristics, and excellent pH buffering capacity;
(4) it should be non-toxic and environmentally friendly; (5) it
should be easily sterilized, manufactured, and handled in the
field, and have good storage qualities; and (6) it should be
inexpensive (Stephens and Rask 2000; Rebah et al. 2002;
Rivera-Cruz et al. 2008).
Organic wastes from animal production and agriculture
and byproducts of agricultural and food processing indus-
tries cause substantial environmental and social problems
in both developed and developing countries. However,
most of these organic wastes meet the requirements of a
biofertilizer carrier. Therefore, they could be good carrier
materials. Perlite is an organic stone of volcanic origin; it
is made of aluminum silicate and contains little water. In
a previous study, this material was a good carrier material
because of its light weight, its porosity, and its environmen-
tal-friendliness (Khavazi et al. 2007). Therefore, in this
study, we evaluated the performance of perlite as a major
component of carrier materials (corn cobs, wheat husks,
and composted cattle manure).
Carrier materials for biofertilizers must meet the criteria
described above, but they also must be sterilized to retain
a large population of the inoculant microorganism during
long-term storage. Gamma-ray irradiation appears to be
a promising sterilization technology because it is easy, it
can be used on a large scale, and it has been shown to
result in reduced storage losses, extended shelf-life, and/
or larger populations of the inoculant during storage (Kha-
vazi et al. 2007; Fernandes et al. 2011). This technology
has been used to sterilize commodities such as tubers and
bulbs, grains, dry ingredients, meat, and fruit (Farkas 2006).
However, gamma-ray irradiation has rarely been used to
sterilize carrier materials for biofertilizers, and its effects on
the attributes of carriers are unknown (Khavazi et al. 2007).
Thus, we evaluated the effects of gamma-ray irradiation and
autoclaving on the ability of the carrier materials to sustain
populations of the inoculant during storage, and on the ability
3. 160 WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167
of the biofertilizer on these carrier materials to colonize soil
and promote plant growth.
The objectives of this study were to select a suitable car-
rier for inoculating PSF, to confirm the sterilization effect of
gamma-ray irradiation during biofertilizer preparation, and
finally to develop an inexpensive and simple P-biofertilizer,
which could be widely produced by most biofertilizer man-
ufacturers in developing countries using currently available
technologies. We evaluated the suitability of carriers con-
sisting of materials that are widely available in developing
countries, including corn cobs, wheat husks, and composted
cattle manure mixed with perlite, in addition to peat. We
compared the shelf-life of P-biofertilizer among the different
carrier materials. The P-biofertilizer was inoculated into
soil in a pot experiment. The survival of the fungus, its
effects on available P in soil, and its effects on the growth
of Chinese cabbage were evaluated. To investigate shelf
life, we compared inoculants stored at room temperature
(25°C) and at 4°C, respectively.
2. Results
2.1. Isolation of P-solubilizing fungi
Based on morphological features, 30 morphotypes of fungi
isolates were recovered. All 30 isolates were screened for
P-solubilizing activity. 13 isolates (including 11 Penicillium
and 2 Aspergillus) with remarkable P-solubilizing activity on
National Botanical Research Institute’s phosphate growth
(NBRIP) agar, as visualized by a clear zone around the
colony, were selected. To identify the strain with the stron-
gest P solubilizing ability, we analyzed the change in pH
and the amount of solubilized P in NBRIP liquid medium.
As shown in Fig. 1, strain 1107, which was identified as
A. niger according to its colony morphology and microscopic
characteristics, caused a decrease in pH from 7.0 on day
0 to pH 3.0 on day 10. After 10 d of culture, this strain had
solubilized and released 689 mg P L–1
(Fig. 1).
2.2. Shelf-life of A. niger strain 1107
We evaluated the survival of A. niger strain 1107 in various
carriers sterilized by autoclaving or gamma-ray irradiation
and stored at 4 and 25°C, respectively for 7 mon. The
survival of A. niger strain 1107 in various carriers sterilized
using autoclaving or gamma-ray irradiation prior to storage
at 4°C is shown in Fig. 2. During the first 5–6 mon of stor-
age, most of the carriers (except composted cattle manure
with 20% perlite, CCMP) sterilized by gamma-ray irradiation
had higher inoculum loads than those in carriers sterilized
by autoclaving. This effect was not observed after 7 mon
of storage.
In the first 2 wk of storage, the number of viable cells
increased dramatically in all of the carrier materials. The
population of A. niger 1107 was higher than 7.1 log CFU
spores g–1
inoculant for at least 7 mon at 4°C. This popula-
tion size met the standard considered as acceptable in most
countries (9 log CFU g–1
inoculant) (Ogbo 2010). The only
exception was the population of A. niger in CCMP, which
decreased from 7.8 to 4.5 log spores g 1
inoculant in 7 mon.
Compared with CCMP, corn cobs with 20% perlite (CCP)
and wheat husk with 20% perlite (WHP) were better mate-
rials to support the growth of, and maintain the population
of, A. niger. The CCMP, CCP, and WHP carriers contained
1.2×107
, 2.0×107
, and 3.2×107
spores g–1
inoculant, respec-
tively, after 7 mon. As shown in Fig. 3, the spore survival
rate was higher in P-biofertilizer stored at 25°C than in that
stored at 4°C during the first 1–3 mon, but after 3 mon, the
survival rate was higher at 4°C than at 25°C.
2.3. Efficacy of biofertilizer
We conducted a pot experiment to evaluate the ability of
A. niger 1107 to colonize soil and its effects on plant growth.
In all of the treatments, the A. niger 1107 population in-
creased in the first 3 wk, and then decreased (Fig. 4). By 21
d after inoculation, the soil samples contained 1.1–6.6×106
spores of A. niger g–1
soil. As time went on, the population
of A. niger 1107 decreased to 0.9–56.2×105
viable spores
before plant harvesting. The most promising phosphate-sol-
ubilizing microorganisms (PSM) biofertilizer in the shelf-life
experiment (that on WHP) showed the highest soil coloni-
zation rate (5.6×106
spores of A. niger g–1
soil), which was
similar to that of the peat-based biofertilizer in soil. These
results indicated that WHP is a good carrier material for
biofertilizer. In contrast, composted cattle manure was
not a suitable biofertilizer carrier, since the A. niger 1107
0 2 4 6 8 10
0
100
200
300
400
500
600
700
Soluble P pH
Inoculation time (d)
SolubeP(mgL−1
)
0
2
4
6
8
pH
Fig. 1 Changes in pH and soluble P concentration during
fermentation in National Botanical Research Institute’s
phosphate growth (NBRIP) liquid medium.
4. 161WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167
spores in this material showed the lowest soil colonization
rate, even lower than that of free A. niger 1107 cells. The
soil colonization experiment showed that the survival of
A. niger strain 1107 in peat, CCP, and WHP sterilized by
gamma-ray irradiation was 3.87, 0.4, and 5.62 log CFU
g–1
inoculant, respectively, and that in the same materials
0 50 100 150 200 250
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0ThepopulationofAspergillusniger1107(logCFUg−1
inoculant)
Irradiated Autoclaved
Peat
0 50 100 150 200 250
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
Corn cop with 20% perlite
0 50 100 150 200 250
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
Storage time (d)
Wheat husk with 20% perlite
0 50 100 150 200 250
3
4
5
6
7
8
9
10
Storage time (d)
Composted cattle manure with 20% perlite
Fig. 2 Survival of A. niger strain 1107 in various carriers sterilized using autoclaving or gamma-ray irradiation prior storage at 4°C.
Vertical bars represent means of triplicates±SD. The same as below.
0 50 100 150 200 250
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
0 50 100 150 200 250
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
0 50 100 150 200 250
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
0 50 100 150 200 250
2
3
4
5
6
7
8
9
10
Storage time (d)
ThepopulationofA.niger1107(logCFUg−1
inoculant)
Peat
Corn cop with 20% perlite
4°C 25°C
Wheat husk with 20% perlite Composted cattle manure with 20% perlite
Storage time (d)
Fig. 3 Survival of A. niger strain 1107 in various carriers sterilized using gamma-ray irradiation when stored at 4 and 25°C.
5. 162 WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167
sterilized by autoclaving was 3.29, 0.2, and 3.16 log CFU
g–1
inoculant, respectively. Therefore, the differences in the
amount of A. niger 1107 spores among different materials
were far greater than the difference in the amount of A. niger
1107 spores between the same carrier sterilized using two
different methods.
We evaluated the beneficial effects of P-biofertilizer on
the growth of Chinese cabbage in a pot experiment. The
effects of A. niger 1107 inoculations as both liquid and solid
inoculants on the length of roots and shoots, wet weight,
and dry weight are shown in Table 1. The root length and
weight (including wet and dry mass) of plants inoculat-
ed with A. niger 1107 were significantly (P<0.05) higher
than those of uninoculated control plants. Treatments with
A. niger 1107 on peat and WHP carriers were significantly
more effective (P<0.05) for promoting plant growth than
those treated with A. niger 1107 as free cells or on the
CCMP carrier. The maximum increases in root length and
dry weight (91.7 and 106.7%, respectively) were in plants
treated with A. niger 1107 on gamma-irradiated peat. There
were also very large increases in root length and dry weight
(83.3 and 93.3%, respectively) in plants treated with A. niger
1107 on gamma-irradiated WHP.
As shown in Table 1, after plant harvest, the amount of
available P in soil treated with the A. niger 1107 was higher
than that in non-inoculated soil. The P content in TCP-sup-
plemented soil increased from 7.5 to 24.3 mg kg–1
after
inoculation with the P-solubilizing fungus. The available P
content differed significantly (P<0.05) among soils treated
with biofertilizer on different carriers, but not between soils
treated with biofertilizer on the same carrier sterilized using
two different methods. The greatest increase in available P
(a 1.6-fold increase) was in soil treated with A. niger 1107
on gamma-irradiated TCP.
3. Discussion
In this work, A. niger strain 1107 showed a strong P-solu-
bilizing ability (689 mg P L–1
), which might be because of
its strong ability to produce organic acids. Other studies
reported that the ability of many fungi to solubilize phos-
phates in vitro is generally associated with the release of
organic acids, which decreases the pH of the growth medium
(EI-Azouni 2008; Ogbo 2010). As reported by Ogbo et al.
(2010), there are wide variations among species in terms
of the amount of soluble phosphate produced in liquid
cultures (9.47–1235 mg L–1
). Even within the same fungal
0 7 14 21 28 35 42 60
0
100
200
300
400
500
600
700
800
ViablesporesofA.Niger
(CFU×104
g−1
)
Incubation time (d)
1107
1107 in γ-irradiated peat 1107 in autoclaved peat
1107 in γ-irradiated CCP 1107 in autoclavd CCP
1107 in γ-irradiated WHP 1107 in autoclaved WHP
1107 in γ-irradiated CCMP 1107 in autoclaved CCMP
Fig. 4 Soil colonization of A. niger strain 1107 in various
carriers sterilized by gamma-irradiation and autoclaving during
plant growth. CCP, corn cob mixed with 20% of perlite; WHP,
wheat husk mixed with 20% of perlite; CCMP, composted cattle
manure mixed with 20% of perlite.
Table 1 Effects of inoculation Aspergillus niger 1107 on growth and soil available P after harvest of Chinese cabbage plants under
pot experiment
Treatment1)
Shoot length
(cm)
Root length
(cm)
Plant weight
(mg plant–1
)
Dry mass
(g plant–1
)
Soil available P after harvest
(mg kg–1
)
Soil 9.5±1.5 a 1.2±0.3 a 42.8±1.8 a 4.5±0.6 a 14.9±0.9 a
Soil+TCP 10.2±1.2 a 1.3±0.3 ab 44.5±1.5 ab 4.6±0.6 a 17.5±1.5 b
Soil+TCP+1107 10.8±1.4 a 1.7±0.2 bc 54.5±1.9 bc 5.8±0.2 b 19.4±0.7 c
Soil+TCP+1107 in γ-irradiated peat 14.9±1.2 cd 2.3±0.3 e 80.4±5.7 e 9.3±0.8 e 24.3±0.7 f
Soil+TCP+1107 in autoclaved peat 14.6±1.0 bcd 2.2±0.5 e 78.1±6.6 e 9.1±1.2 e 23.8±1.0 f
Soil+TCP+1107 in γ-irradiated CCP 13.5±0.8 bc 2.0±0.3 cde 71.3±4.6 de 8.0±0.7 cd 20.5±0.9 cd
Soil+TCP+1107 in autoclaved CCP 13.1±1.2 b 1.8±0.4 bcd 70.7±7.8 d 7.8±0.3 c 19.4±1.0 c
Soil+TCP+1107 in γ-irradiated WHP 14.6±1.0 bcd 2.1±0.3 de 75.2±6.0 de 8.5±0.8 cde 21.8±0.8 de
Soil+TCP+1107 in autoclaved WHP 15.4±0.8 d 2.2±0.5 e 76.1±5.7 de 8.7±0.4 de 22.2±0.8 e
Soil+TCP+1107 in γ-irradiated CCMP 11.7±1.5 a 1.7±0.3 bcd 55.2±4.3 c 6.2±0.3 b 20.0±1.5 c
Soil+TCP+1107 in autoclaved CCMP 11.5±1.3 a 1.6±0.2 abc 53.4±4.8 c 6.0±0.5 b 19.8±1.2 c
1)
TCP, tri-calcium phosphate; CCP, corn cob mixed with 20% of perlite; WHP, wheat husk mixed with 20% of perlite; CCMP, composted
cattle manure mixed with 20% of perlite.
Results represent the mean of three plot (five plants in one plot) replicates±SD. Different letters show values that are significantly
different (P<0.05) according to the Duncan’s test.
6. 163WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167
species, the wide differences in experimental conditions
used in various studies may explain some of the variations
in P-solubilization ability, but the P-solubilization ability also
varies markedly among different organisms and strains
(Lapeyries et al. 1991).
Previous works indicated that methods used to sterilize
various carriers can substantially affect microorganism
survival (Strijdom and Van Rensburg 1981; Khavazi et al.
2007). Similarly, Khavazi et al. (2007) reported that rhi-
zobial populations were larger in materials pre-sterilized
by gamma-ray irradiation than in those pre-sterilized by
autoclaving. After 6 mon, however, this effect was only
significant for a perlite/sugarcane bagasse mixture. In
the shelf-life experiments, the survival of A. niger strain
1107 in the carrier materials sterilized by autoclaving was
approximately 2 to 4 times higher than its survival in those
sterilized by irradiation. This is probably because of chang-
es in the chemical composition of the carriers during the
autoclaving process (Strijdom and Van Rensburg 1981).
Irradiation of the carrier material should not cause any
physical and chemical changes to the inoculant, or cause
it to produce toxins (Parker and Vincent 1981; Rizzuti
et al. 1996; Daza et al. 2000). In the present study, the
advantage of gamma-ray irradiation over autoclaving was
not detected after 7 mon of storage. This may be because
spore-forming or cyst-forming microorganisms were able to
recover and replicate after sterilization with irradiation, and
therefore, compete with the inoculant (Yardin et al. 2000).
Gamma-ray irradiation is a good sterilization treatment
because it is easy and it can be used on a large scale.
However, it is important to note that gamma-ray sterilizers
are not readily accessible in less developed countries.
The storage temperature is another factor that affects the
shelf-life of biofertilizers. Accinelli et al. (2009) reported
a greater decline in A. flavus NRRL 30793 propagules at
25°C than at 4°C during a 6-mon storage period. Consis-
tent with this, our results showed that room temperature
was suitable for a short storage period, but 4°C was better
for prolonging shelf-life during long-term storage.
Generally, the biofertilizer on carriers promoted plant
growth more effectively than did the free-cell biofertilizer.
This is because carriers protect functional microbes from
soil or climatic stresses (Daza et al. 2000; Jain et al. 2010).
Carrier materials may enhance the survival of inocula by
providing microorganisms with a protective environment.
This can allow them to survive in unfavorable conditions
during the preservation and soil colonization process. In
particular, once the microbe is introduced into soil, it must
be able to survive in the subsurface zone to effectively sol-
ubilize P independently of the ecological conditions. There
are many instances where different carrier materials have
improved biofertilizer growth and survival. Finely ground
peat is the most commonly used carrier in conventional
inoculant production. However, peat is not always available,
and the need to preserve wetland ecosystems makes the
extraction of peat inadvisable in some areas. Thus, there is
a need to identify other materials that support good growth
and survival of microorganisms. Agro-wastes are good
alternatives to peat as carrier materials. Ogbo (2010) used
cassava wastes as carriers for two fungi, A. fumigatus and
A. niger. In that study, the ground cassava waste met many
of the criteria for a carrier material, and it supported the
growth of the studied organisms. Rivera-Cruz et al. (2008)
used poultry manure and banana waste as the inoculant
carrier for P-solubilizing bacteria. They found that the
application of these biofertilizers improved plant perfor-
mance and the physical and microbiological properties of
the soil. Perlite has been used as a carrier for Rhizobium
leguminosarum bv. phaseoli, R. tropici, Bradyrhizobium
japonicum, and Bacillus megaterium inoculants. The
viable colony counts of these bacteria remained stable at
107
–109
g–1
for at least 180 d when the carrier was stored
at 4 or 28°C (Daza et al. 2000). Six different mixtures of
perlite and charcoal/sugarcane bagasse were evaluated
as carriers for Bradyrhizobium japonicum, and all of them
supported rhizobial growth over a 6-mon period (Khavazi
et al. 2007). In the present study, populations of the P-sol-
ubilizer A. niger remained stable in sterilized WHP for at
least 7 mon. This is because of its high nutrient content,
high water holding capacity, and good aeration character-
istics (Table 2), which are three key characteristics of good
carriers according to Smith (1992). Our results showed that
composted cattle manure was not suitable as biofertilizer
carrier to enhance the microbial population. This might be
Table 2 Chemical and physical characteristics of materials used as carriers
Material N (%) P (%) K (%) EC (ds m–1
)1)
pH OM (%)2)
WHC (%)3)
Peat 1.73 0.21 0.32 0.24 6.14 65.4 100
Wheat husk 0.42 0.15 1.14 2.08 5.43 31.5 300
Corn cob 0.35 0.06 0.91 1.07 5.64 89.4 420
Composted cattle manure 1.63 1.03 1.78 24.5 5.50 46.2 120
1)
EC, electrical conductivity.
2)
OM, organic matter.
3)
WHC, water holding capacity.
7. 164 WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167
because of its higher soluble salt content (EC 24.5 dS m–1
),
which would affect major microbial processes including
respiration and ammonification. It has been reported that
salt-tolerant fungi, actinomycetes, and a few bacteria can
survive at higher salt concentrations (Rynk 1992; EI-Azouni
2008). In general, EC values between 0 and 3.5 dS m–1
are
acceptable for general crop growth; at EC values exceeding
that range, the salt concentration will hinder plant growth by
affecting the soil-water balance (Naidu et al. 2010).
As shown in Fig. 4, the soil colonization experiment
showed that the differences in the survival counts of
A. niger strain 1107 among the various carriers sterilized by
different methods ranged from 117 to 235%. In contrast,
the differences in survival counts among the various carrier
materials ranged from 145 to 6244%. It is noteworthy that
compared with the complex soil environment, the effect of
the sterilization method on the viability of A. niger strain 1107
spores during plant growth was insignificant. This result
indicated that the changes to the carriers resulting from the
sterilization procedure were negligible. The wide range in
survival counts among the different carrier materials showed
that some materials were much more suitable than others
for survival of A. niger strain 1107. After 60 d incubation,
the highest survival count was in peat, followed by WHP.
Consistent with our findings, inoculation with P solubiliz-
ers has been reported to increase the soil available P content
(Mittal et al. 2008; Jain et al. 2010; Jain et al. 2012). The
effects of A. niger 1107 to promote plant growth may be relat-
ed to the increase in available phosphate in soil. This would
also benefit the next crop, as more available phosphate in
soil would increase its fertility (Jain et al. 2010). In summary,
although WHP was not superior to peat as a carrier, it is
a suitable alternative carrier to peat for the P-solubilizing
strain of A. niger. These results are important because the
demand for biofertilizer in China is greatly increasing, but
biotechnological resources, especially carrier materials for
biofertilizer, are limited.
4. Conclusion
According to the shelf-life experiment, gamma-ray irra-
diation is suitable as a simple sterilization method that
can be used to treat large amounts of material. The most
effective carrier material for growth and maintenance of
PSF populations was WHP; there was a higher rate of
conidia survival on this material than on other materials,
and when it was applied as a biofertilizer to Chinese
cabbage, it beneficially affected plant yield and increased
the soil available P concentration. The wide application
of P-biofertilizer as an alternative to chemical fertilizers in
developing countries will contribute to lower-input farming
systems and a cleaner environment.
5. Materials and methods
5.1. PSM isolation and identification
More than 20 rhizospheric soil samples were collected
from various farmlands in Beijing, Hebei Province, and
Heilongjiang Province, China. For each sample, 1 g soil was
suspended in 10 mL sterilized water, diluted to 10–7
, and then
100 µL of each dilution was spread on the potato-dextrose
agar (PDA) medium. After incubation of the plates for 4 d
at 30°C, colonies on the plates were selected, purified by
repeated culturing, and maintained on PDA slants at 4°C.
Preliminary screening for phosphate solubilization was
conducted by a plate assay method using modified National
Botanical Research Institute’s phosphate growth (NBRIP)
agar medium (pH 7.0) (Nautiyal 1999). The medium
contained (in g L–1
): glucose, 10.0; Ca3
(PO3
)2
,5.0; (NH4
)2
SO4
,
0.5; NaCl, 0.3; KCl, 0.3; MgSO4
·7H2
O, 0.3; FeSO4
·7H2
O,
0.03; MnSO4
·4H2
O, 0.03; and agar, 18. Fungal strains
were pin-point inoculated onto plates containing solidified
NBRIP medium under aseptic conditions. The plates were
incubated at (28±2)°C for 7 d and the diameter of fungal
colonies was continuously observed. The fungal colonies
surrounded by a clear halo zone showed P-solubilization
activity, and were selected for further tests. These strains
were maintained on PDA slants at 4°C. To confirm the
P-solubilizing activities of the selected strains, each strain
was incubated in 100 mL NBRIP liquid medium at 30°C and
120 r min–1
on a rotary incubator shaker for 10 d, and then the
amount of P released was measured by the molybdenum-
blue method (Murphy and Riley 1962). At the same time,
pH was recorded using electrode pH meter (Mettler Toledo
Delta 320).
5.2. Carrier preparation
Carrier materials were first powdered and passed through
a 100-mesh sieve before physicochemical characterization
(Table 2). The main criteria used to select carrier materials
were the ability to adjust the pH to neutral (pH 7.0), high
water holding capacity, low cost, and wide availability. The
pH of all of the materials was adjusted to pH 7.0 with CaCO3
before use. To adjust pH, materials were thoroughly mixed
with CaCO3
powder. Four carriers: (1) peat, (2) corn cob
mixed with 20% perlite (CCP), (3) wheat husk mixed with
20% perlite (WHP), and (4) composted cattle manure mixed
with 20% perlite (CCMP) were evaluated.
5.3. Carrier sterilization
For each of the four carriers, 500 g of carrier material was
8. 165WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167
placed into each of 10 cotton bags (50 cm×16 cm). The
cotton bags were approximately 1.0 mm thick. These
packages were sterilized by gamma-ray irradiation or
autoclaving.
For irradiation, the packages were placed in 0.08-mm-
thick polypropylene plastic bags. Gamma-ray irradiation
was carried out using a 60
Co gamma cell source. Irradiation
was applied at a dose of 50 kGy at a rate of 15 Gy min–1
.
For the autoclaving treatment, the samples were
autoclaved for 40 min at 121°C. The sterilized bags were
placed into another large sterile cotton bag after cooling
overnight in the autoclave. After sterilization, the packages
were dried for 12 h at 60°C in a blow-type oven.
5.4. Inoculant preparation and incubation
To obtain fresh spores of strain 1107, the fungus was
grown on NBRIP agar medium at 30°C for 5 d. The spores
were recovered from plates in 5 mL sterile 0.2% Tween-20
collected by gentle scraping. The concentration of spores was
determined by microscopic counts using a hemocytometer.
The spore density was adjusted to a final concentration of
108
spores mL–1
.
The spore suspension was injected aseptically into the
sterilized carriers to 40% of water holding capacity using
a sterile syringe injector. The bags were then thoroughly
kneaded and incubated at 30°C for 2 wk before the storage
experiment. The population size of strain 1107 in the carriers
was measured by the plate count method.
5.5. Determination of shelf-life
To evaluate shelf-life, the formulated products were
transferred into sterilized screw-top tubes (50-mL volume)
and stored in the dark at 4 and 25°C. The biofertilizer was
turned over every 3-4 d for 7 mon. Non-inoculated raw
material was used as the control. After 7, 14, 30, 60, 90,
120, 150, 180, 210 d of storage, the amount of surviving
fungus (spores) was evaluated by the plate count method.
Aseptic conditions were maintained throughout this process.
5.6. Pot experiment
Black soil (top soil, 0–20 cm) was collected from an arable
field in Heilongjiang Province, China. Air-dried soil samples
were ground and homogenized by passing through a 100-
mesh sieve after removing stones, soil fauna, and plant
debris. The soil properties were as follows (g kg–1
air-dry
soil): sand (0.05–0.2 mm), 280; silt (0.002–0.05 mm), 450;
clay (<0.002 mm), 270; organic carbon, 23.03; total N, 1.28;
available P 0.02; and pH 5.7 (1:2.5 soil/water mixture).
A mixture of 0.5 g tri-calcium phosphate (TCP) per kilogram
soil was prepared and placed in plastic pots (height, 13 cm;
upper diameter, 12 cm; lower diameter, 9 cm). The pots with
different treatments were arranged in a randomized complete
block design with triplicates of each treatment. There were
11 treatments in total: T1: soil; T2: soil+TCP; T3: soil+TCP+
A. niger 1107; T4: soil+TCP+A. niger 1107 in peat sterilized
by gamma-ray irradiation; T5: soil+TCP+A. niger 1107 in peat
sterilized by autoclaving; T6: soil+TCP+A. niger 1107 in CCP
sterilizedbygamma-rayirradiation;T7:soil+TCP+A.niger1107
in CCP sterilized by autoclaving; T8: soil+TCP+A. niger 1107
in WHP sterilized by gamma-ray irradiation; T9: soil+TCP+
A.niger1107inWHPsterilizedbyautoclaving;T10:soil+TCP+
A. niger 1107 in CCMP sterilized by gamma-ray irradiation;
T11: soil+TCP+A. niger 1107 in CCMP sterilized by
autoclaving.
Chinese cabbage seeds were surface sterilized by
immersion in 0.1% sodium hypochlorite solution for 10 min.
The seeds were washed three times with distilled water
and then planted in pots (9 seeds pot–1
, with equal distance
between seeds). Then, 10 mL spore suspension or 10 g
prepared biofertilizer was applied uniformly to seeds, which
were then covered with a 20-mm-thick soil layer. After 1 h,
the pots were sprinkled with water. The pots were irrigated
periodically to maintain soil moisture at between 30 and 50%.
At 1 wk after germination, the seedlings were thinned to five
per pot. Plants were harvested at 60 d after sowing, and their
shoot and root length and dry mass were recorded. The dry
weight of plant tissues was determined after drying at 75°C.
After harvesting, the available P concentration in pot soil was
analyzed by the NaHCO3
-extractable phosphorus colorimetric
method (Olsen et al. 1954). At various time points (7, 14,
21, 28, 35, 42, 60 d), triplicate soil samples around the seed
were collected and the A. niger 1107 colonization rate was
estimated by the plate count method.
5.7. Statistical analysis
All experiments were conducted in triplicate and data
were subjected to analysis of variance. Mean values
were compared using one-way ANOVA Duncan’s test and
significant differences were detected at the P=0.05 level.
The number of fungal spores derived from soil or inoculant
is expressed as the log(10) transformed colony forming
units (CFU) g–1
weight.
Acknowledgements
This work was financially supported by the Special Fund
for Agro-Scientific Research in the Public Interest, China
(201003014) and the Central Public-Interest Scientific
9. 166 WANG Hong-yuan et al. Journal of Integrative Agriculture 2015, 14(1): 158–167
Institution Basal Research Fund, China (202-27).
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