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OPEN Bio-organic fertilizer with reduced

rates of chemical fertilization
improves soil fertility and enhances
tomato yield and quality
Lin Ye1,2*, Xia Zhao1,2, Encai Bao3, Jianshe Li2, Zhirong Zou1* & Kai Cao3

The extensive use of chemical fertilizers poses serious collateral problems such as environmental
pollution, pest resistance development and food safety decline. Researches focused on applying
plant-beneficial microorganisms to partially replace chemical fertilizer use is increasing due to the
requirement of sustainable agriculture development. Thus to investigate the possibility of a plant-
beneficial Trichoderma strain and its bio-organic fertilizer product in saving chemical fertilizer
application and in improving crop quality, a field trial and continuous pot experiments were carried
out with tomato. Four treatments were set up: a reduced application of chemical fertilizer (75% of the
conventional application) plus Trichoderma-enriched bio-organic fertilizer (BF), organic fertilizer (OF) or
Trichoderma spore suspension (SS), with using the 100% rate of the conventional chemical fertilizer as
the control (CF). The results showed that the total soluble sugar, Vitamin C and nitrate accumulations
were, respectively, +up to 24%, +up to 57% and –up to 62% in the tomatoes of the BF treatment
compared to those of the control (CF). And both of the pot and field trials revealed that reduced rates of
chemical fertilizer plus bio-organic fertilizer produced tomato yields equivalent to those obtained using
the 100% of the chemical fertilizer. However, application with the inoculant alone (SS) or combined with
the organic fertilizer alone (OF) would lead to a yield decreases of 6–38% and 9–35% over the control.
Since the increased abundance of soil microflora and the enhanced soil fertility frequently showed
positive linear correlations especially in the BF-treated soils, we conclude that the efficacy of this bio-
organic fertilizer for maintaining a stable tomato yield and improving tomato quality may be due to the
improved soil microbial activity. Thus, the results suggest that the Trichoderma bio-organic fertilizer
could be employed in combination with the appropriate rates of chemical fertilizers to get maximum
benefits regarding yield, quality and fertilizer savings.

Application of chemical fertilizers is one of the most adopted regimes in developing intensive agriculture now-
days1,2. However, the continuous long-term use of chemical fertilizer has led to many unexpected effects. For
example, the productivity-cost does not scale linearly and results in a huge waste of mineral resources. In addi-
tion, millions of tons of synthetic nutrients that are loaded to soil yearly are not absorbed by plants. About up to
50% of N and 90% of P have been reported to run off from crop fields and escape into the atmosphere or water
sources, thereby causing the generation of greenhouse gas, eutrophication in aquatic system and salinization
in soil2,3. Besides, the excessive application of chemical fertilizer leads to food safety and quality decline prob-
lems, such as nitrate accumulation in vegetal products. Indeed, several studies have demonstrated that organic
farming, which strictly prohibits synthetic fertilizers, provides an alternative that has the potential to minimize
the negative influence from by using chemical fertilization, and the products from the organic farming systems
are generally endowed with improved nutritional properties4–7. For example, the work of Caris-Veyrat et al.4
reported that organic tomatoes had higher contents of carotenoids, polyphenols and Vitamin C than the ones
from the conventional farming agriculture. However, organic farming is always associated with lower yield of

1
Horticulture College, Northwest A&F University, Yangling, Shanxi, China. 2Agriculture College, Ningxia University,
Yinchuan, Ningxia, China. 3The Agriculture Ministry Key Laboratory of Agricultural Engineering in the Middle and
Lower Reaches of Yangtze River, Institute of Agricultural Facilities and Equipment, Jiangsu Academy of Agricultural
Sciences, Nanjing, China. *email: yelin.3993@163.com; zouzhirong2005@hotmail.com

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Pot Experiments (kg pot−1) Field Trial

Treatments a
1st Season 2nd Season 3rd Season 4th Season (kg plot−1)
CF 0.74 ± 0.06a 0.22 ± 0.01b 0.55 ± 0.01a 0.23 ± 0.01a 82.85 ± 3.71ab
BF 0.77 ± 0.13a 0.28 ± 0.02a 0.55 ± 0.01ab 0.25 ± 0.01a 86.78 ± 8.76a
OF 0.68 ± 0.05a 0.16 ± 0.01c 0.52 ± 0.01b 0.18 ± 0.00b 70.51 ± 6.76b
SS 0.68 ± 0.12a 0.18 ± 0.01c 0.44 ± 0.03c 0.17 ± 0.01b —

Table 1. Effect of different treatments on tomato yield in pot experiments and field trials. aCF: 100% chemical
fertilizer; BF: 75% chemical fertilizer + bio-organic fertilizer; OF: 75% chemical fertilizer + organic fertilizer; SS:
75% chemical fertilizer + spore suspension. The mean value ± standard deviation (n = 5). Values with the same
letter do not differ significantly (P < 0.05).

crops and thus a higher cost. Therefore, the use of chemical fertilizers is not able to be eliminated once a consid-
erable food production is expected1,8. Another current proposition for solving the agro-environmental problem
is the integrated nutrient management that does not aim to entirely remove synthetic fertilizers in the near future
instead suggests using microbial inoculations to reduce the amount of fertilizers applied1. Previous studies have
shown that the inefficiency of synthetic fertilizers can be alleviated by applying plant growth-promoting microbes
(PGPM), which are capable of increasing nutrient use efficiency and plant growth while reducing fertilizer inputs
by up to 50% without causing any yield loss relative to the fully fertilized controls2,9,10.
PGPM exert considerable beneficial effects on the growth and development of plants. Many microbial gen-
era have been commercially applied in agriculture11,12. For example, the plant-beneficial effect of Trichoderma
harzianum has been widely studied and some of the strains are commercially applied to fields in China12,13. They
have been previously shown with significant effects on plant growth, root development, biocontrol of soil-borne
diseases and inducing plant systemic resistance12–14. To effectively apply such PGPM, it was suggested to enrich
the strain in some organic substrate (e.g., as the form of bio-organic fertilizer) and then apply, since T. harzianum
was reported to survive better in the rhizosphere after enriched in organic substrates13,15,16. For nearly all com-
mercial uses of Trichoderma for biocontrol and plant growth and yield enhancement, a better understanding of
the interactions among the Trichoderma, the fertilizer, the soil and the plant is essential. Although the number of
research papers focused on reducing the inputs of fertilizer through different PGPM inoculations has increased
in recent years, there are few reports concerning the combination of Trichoderma-enriched bio-organic fertilizer
and reduced chemical fertilizer, especially in a long-term follow-up study, or concerning its potential effect on
food quality16–18. Until recently, the focus has primarily been on the yield rather than the gustative or micro nutri-
tional quality of fresh vegetal products. It could be acceptable for staple foods, but, as far as vegetables and fruits
have been concerned, it can be argued the food quality matters more than the energy supply4. Tomato (Solanum
lycopersicum), one of the most popular vegetables worldwide, is cultivated in over 140 countries19. It has many
metabolites responsible for health and nutritional values, thus making it as an excellent model for studying fruit
development and food safety20. However, in spite of the importance of vegetable quality to human health, there
has been little improvement in the tomato fruit quality since producers preferentially select cultivars with traits
of resistance to biotic and abiotic stress, extended shelf-life and appearance19. And the understanding of how
fertilization regimes influence tomato quality stay poor.
Therefore, the aims of the present research were to investigate 1) if PGPM inoculants (Trichoderma or
Trichoderma bio-organic fertilizer) combined with reduced rates of chemical fertilizer would produce tomato
yield and fruit quality equivalent to those obtained using full rates of chemical fertilizer, and 2) how the soil
fertility responses to these fertilization regimes in a continuous cropping system. We therefore investigated the
effectiveness of a Trichoderma bio-organic fertilizer on tomato yield and quality in an open field and evaluated the
effect of Trichoderma inoculation on some chemical and biological properties of the soil related to plant growth
via continuous 4-season pot experiments. Due to the importance of soluble sugars to a tomato’s organoleptic
quality as well as that of Vitamin C (Vc) to nutrition and nitrate accumulation to safety, it is acceptable to use these
three indexes to indicate the quality of a tomato fruit7,19,21,22. The introduction of a PGPM agent and fertilization
could influence the soil’s microbial diversity and nutrient status and could subsequently change the soil quality;
thus, the soil microbial population and the soil total and available nutrients were measured at the end of each
growing season. In addition, strains with the potential of promoting plant growth and providing other benefits
must be able to colonize roots23; therefore, the number of Trichoderma in the rhizosphere soil was determined in
this study.

Results
Tomato yield. The results (Table 1) of both field trials and pot experiments indicated that the tomato yields
from treatments with 75% chemical fertilizer plus bio-organic fertilizer (BF) were statistically equivalent to the
yields obtained using full rate of chemical fertilizer (CF) without inoculants, while the yields obtained using
75% chemical fertilizer with Trichoderma spore suspension (SS) or organic fertilizer (OF) were less than those of
the CF treatment in most cases significantly (P < 0.05) (Table 1). In contrast to the control (CF), the OF and SS
treatments led to a decrease of 6–38% and 9–35%, respectively, in tomato yield in the pot experiments. The results
were slightly different among the 4 pot experiments as the yields of the 4 treatments in the 1st season did not
differ from each other. Because the data of the 1st field trial were more persuasive than those from the 2nd trial,
here and similarly hereafter, only the data of the 1st field trial are shown.

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Figure 1. Effects of different treatments on tomato fruit quality in the open field. CF: 100% chemical fertilizer;
BF: 75% chemical fertilizer + bio-organic fertilizer; OF: 75% chemical fertilizer + organic fertilizer. Error
bars represent the standard deviation calculated from 5 replicates. Bars followed by the same letter are not
significantly different at P < 0.05.

Tomato quality parameters. The results in Fig. 1 showed that, in field trials, the fertilization treatments
had significant (P < 0.05) effect on the total soluble sugars (TSS) content and NO3- accumulation in tomato fruit
while had little effect on Vc content. Specifically, TSS content was approximately 40% higher in fruit from the BF
or OF treatments than it was in fruit from the control (CF), whereas the NO3- content in fruit from the CF treat-
ment was approximately 70% higher than that in fruit from the reduced chemical fertilization treatments (BF and
OF). The results in Fig. 2 showed that different fertilization significantly affected the quality of the tomato fruit
in the pot experiments. The variance in the Vc content observed in the tomato fruit in the 4 treatments was not
obvious in the 1st and 3rd seasons, but it was evident in the 4th season: specifically, the Vc contents in the organic
material added treatments (BF and OF) were 23–30% higher than the Vc contents in the synthetic fertilizer treat-
ments (CF and SS) (Fig. 2a). The chromatograms of Vc and NO3- analysis are illustrated with Figs. S1 and S2. The
TSS contents in the four treatments gradually increased with continuous cropping, and the NO3- contents gen-
erally increased first and subsequently decreased; the TSS content in the 3 reduced chemical fertilizer treatments
(BF, OF and SS) were much higher than with the full rate chemical fertilizer treatment (CF), while the trend was
opposite in NO3- accumulation throughout the 4 cropping seasons (Fig. 2b,c).

Soil nutrients content and organic matter. In general, the soil available nutrient contents at the end
of the growing season reflect the soil nutrient supply. In the field trial, the available P and available K in the BF
treatment were significantly higher than the contents in CF and OF treatments (P < 0.05) (Fig. 3). Conversely, no
significant differences were observed in soil nitrate-N among the 3 treatments, and the ammonium-N in the CF
treatment was markedly higher than the contents in the BF and OF treatments.
Figure 4 showed the fluctuations in available nutrients over time in the pot experiments. A similar trend of
available P and available K was found in both the pot experiments and the field trials. The available P and avail-
able K in the organic material added treatments (BF and OF) were significantly higher than the contents in the
synthetic fertilizer treatments (CF and SS), while the ammonium-N in the CF treatment was the highest among
the 4 treatments throughout the 4 copping seasons (Fig. 4a,c,d). The soil nitrate-N gradually grew with time, and
its content in the BF and OF treatments remained at a higher level compared with that of the CF (Fig. 4b). Higher
levels of nitrate-N, available P and available K were more frequently observed in the BF treatment, whereas the
lowest contents of available nutrients, including all of the above indexes, were mostly found in the SS treatment.
The data in Fig. 5 showed that bio-organic or organic fertilization (BF and OF) significantly enriched soil
fertility, as the soil organic matter increased by 55–75%, total N by 25–36%, total P by 116–123% and total K by
99%–100% after the 4-season fertilization. Total P and total K also improved following the CF and SS treatments
(Fig. 5c,d), while the soil organic matter maintained a stable level, and the total N decreased with time in these 2
treatments (Fig. 5a,b). Higher levels of total nutrients were always found in the OF treatment.

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Figure 2. Effects of different treatments on tomato fruit quality with cropping seasons in the greenhouse.
CF: 100% chemical fertilizer; BF: 75% chemical fertilizer + bio-organic fertilizer; OF: 75% chemical
fertilizer + organic fertilizer; SS: 75% chemical fertilizer + spore suspension. Error bars represent the standard
deviation calculated from 5 replicates.

Figure 3. Effects of different treatments on soil available nutrients in the open field. CF: 100% chemical
fertilizer; BF: 75% chemical fertilizer + bio-organic fertilizer; OF: 75% chemical fertilizer + organic fertilizer.
Error bars represent the standard deviation calculated from 5 replicates. Bars followed by the same letter are not
significantly different at P < 0.05.

Soil bacteria, fungi, actinomycetes and Trichoderma population. The colony forming unites (cfu)
of culturable soil microbes including bacteria, fungi and actinomycetes and Trichoderma, were transformed into
their logarithms, as shown in Fig. 6. The total bacteria population of the pot soils in the BF, OF and SS treat-
ments increased (to Log8.6, Log8.5 and Log8.2, respectively), while the number in the CF treatment decreased
(to Log7.9) slightly (Fig. 6a). The total fungi population generally first decreased, then increased and divided
into 2 groups (one group including the BF and OF treatments decreased, and the other including the CF and SS
treatments kept increasing) (Fig. 6b). The total actinomycetes population for all of the treatments first showed a
decrease and then an increase (Fig. 6c). The Trichoderma population in the soil generally first increased and then
decreased in the BF and SS treatments, whereas it remained at a low level in the CF and OF treatments, which
were non-inoculated (Fig. 6d). Additionally, the microbial population in the BF treatment always maintained
the highest level among the 4 treatments, while the CF treatment was more frequently found to have the lowest
microbial population.

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Figure 4. Effects of different treatments on soil available nutrients with cropping seasons in the greenhouse.
(a) Soil ammonium-N; (b) Soil nitrate-N; (c) Soil available P; (d) Soil available K. CF: 100% chemical fertilizer;
BF: 75% chemical fertilizer + bio-organic fertilizer; OF: 75% chemical fertilizer + organic fertilizer; SS: 75%
chemical fertilizer + spore suspension. Error bars represent the standard deviation calculated from 3 replicates.

Pearson correlations of soil nutrients and microbial abundance with tomato yield and quality.
In total, 120 correlations among the soil nutrients, microflora, tomato yield and fruit quality properties were
detected (Table 2). Of these correlations, 19 and 28 correlations had a significance level of 0.05 and 0.01, respec-
tively. In particular, there was no correlation between the tomato fruit yield and the other parameters detected.
The Vc content in the tomato fruit was found to have a positive correlation with soil organic matter but a negative
correlation with NO3- accumulation in fruit. The TSS content in the fruit was positively correlated with all of the
soil parameters detected in this work, with the exception of a negative correlation with the soil ammonium-N.
The NO3- content in the fruit was negatively correlated with several soil parameters, including total N, organic
matter and the bacteria population, and positively correlated with ammonium-N. The Trichoderma population
in soil showed positive internal links with the soil fungi and actinomycetes populations, as well as a negative
link with the soil ammonium-N. Furthermore, 2 major correlation groups with numerous internal links were
observed. The first group consisted of strong positive links among the soil nutrient parameters themselves,
especially the strong positive links between the organic matter and the other nutrient parameters. The second
group included connections (some positive and some negative) among the soil nutrient parameters and the soil
microflora.

Discussion
The reduced load of fertilizers into crop fields without causing productivity lost is a feasible but difficult chal-
lenge2. The results of the field trials confirmed our hypothesis that the combination of Trichoderma-enriched
bio-organic fertilizer with reduced (75%) chemical fertilizers (BF) could produce tomato yield equivalent to or
higher than those obtained using full rates of the fertilizer (CF) (Table 1). This result was consistent with repeated
observations in pot experiments. However, inoculation with the Trichoderma alone (SS, without organic substrate
supply) or supplement with organic fertilizer alone (OF, without Trichoderma) would cause 6–38% and 9–35%
decreases in yield over the control (CF) in the presence of 100% chemical fertilizer doses. Hence, only bio-organic
fertilizer could be a viable supplementary strategy to maintain or increase tomato yields. Similar results regarding

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Figure 5. Effects of different treatments on soil total nutrients with cropping seasons in the greenhouse. (a)
Soil organic matter; (b) Soil total N; (c) Soil total P; (d) Soil total K. CF: 100% chemical fertilizer; BF: 75%
chemical fertilizer + bio-organic fertilizer; OF: 75% chemical fertilizer + organic fertilizer; SS: 75% chemical
fertilizer + spore suspension. The standard deviation (n = 3) was analyzed using a one-way ANOVA.

PGPM application was also reported by Adesemoye et al.11 that the growth and yield of tomato grown in a green-
house with 75% fertilizer plus PGPM were statistically equivalent to those obtained using full fertilizer rates
without PGPM. Moreover, some reports showed that bio-organic fertilizers could replace 23–52% of N fertilizer
without any loss of yield24, which also indicating the potential role of bio-organic fertilizer in reducing the amount
of chemical fertilizer load to soil. Whereas, few previously reported evidences regarding the influence of PGPM or
its bio-fertilizer application on crop quality could be found8,11. The effects of the same treatment on the measured
yield differed greatly throughout the four growing seasons may due to the variations of climatic conditions.
In this study, at reduced fertilizer rates (75%), inoculants consistently enhanced tomato fruit quality (Figs. 1
and 2). In particular, NO3- accumulation in fruit, which is associated with risks of several human health prob-
lems such as methemoglobinemia and gastric cancer22, from the reduced chemical fertilization (BF, OF and
SS) was coupled with a lower (down to 62%) content compared with the full-rate fertilized control (CF) in the
greenhouse. Actually, NO3- is relatively non-toxic, but its metabolite products, especially NO2-, raised concern
in human health25. As 85–90% of an adult dietary intake of NO3- is from vegetables, while the vegetables tend to
concentrate NO3- when grown in a N highly applied field22,26. Therefore, a proper control of the application rate
of chemical fertilizers to soil would directly reduce our risks to some nitrate-related health problems. Moreover,
the Vc and TSS contents in the tomato fruit in the treatments with added organic material (BF and OF) were sig-
nificantly higher (up to 24% and 57%, respectively) than that of the control (CF). As suggested by Oliveira1 et al.7,
organic farming creates stressing conditions for tomatoes and thus resulted in the higher accumulation of soluble
solids in fruits such as sugars and other compounds that contribute to the nutritional quality of fruit, including Vc
and phenolic compounds. The differences observed in tomato yield and quality for different treatments may orig-
inate from the differences in nitrogen availability because the organic fertilizers (including bio-organic fertilizer
and organic fertilizer) release N (more obviously as ammonium-N in this study; Figs. 3 and 4) much more slowly
than do chemical fertilizers, thus forcing the plants to struggle more for growth and resulting in a more complex
diversion of protein synthesis and secondary metabolisms4,27.
Concerning the other elements, more available P and available K were found in the BF- and OF-treated soils
at the end of each growing season. T. harzianum has been reported to be able to solubilize several plant nutri-
ents28, and T. asperellum has been shown to enhance the P and Fe availability for cucumber once colonized on
roots29. However, the enhancement of available K in the BF treatment in this study did not occur via K dis-
solution because T7 has shown no direct K dissolution ability in vitro under laboratory conditions (data not
shown). Hence, the resulted enhancement of K availability must be due to alternative microbial effects. As shown
in Fig. 6, the treatment with reduced chemical fertilization plus Trichoderma bio-organic fertilizer (BF) resulted

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Figure 6. Effects of different treatments on microbial abundance with cropping seasons in the greenhouse.
(a) Total population of cultivable bacteria in pot soil; (b) Total population of cultivable fungi in pot soil; (c)
Total population of cultivable actinomycetes in pot soil; (d) Total population of cultivable Trichoderma in
pot soil. CF: 100% chemical fertilizer; BF: 75% chemical fertilizer + bio-organic fertilizer; OF: 75% chemical
fertilizer + organic fertilizer; SS: 75% chemical fertilizer + spore suspension. Quantification of the total cultivable
microorganisms in the soils was performed using the standard 10-fold dilution plating method via colony
forming units (cfu) and expressed as its logarithm. The data fulfilled the prerequisites of one-way ANOVA.

in a greater increase of the population of bacteria, fungi and actinomycetes relative to the other treatments. Thus,
the BF treatment was a more efficient tool to enhance the abundance of soil microflora, which had a potential
effect on soil nutrient cycling. Based on the explanations by Adesemoye et al.11 and Huang et al.30, T. harzianum
promotes plant root growth via the secretion of an auxin-like phytohormone. Better root growth impacts soil
microbiome via secreting more exudates, like sugars and organic acids, and soil microbes, in turn, response with
higher microbial abundance and frequent interactions leading to more available nutrients that affect plant growth
and health. Gradually, better plant growth caused by more bio-available nutrients again stimulates more microbes
moving towards the rhizosphere resulting in higher nutrient uptake by the inoculated plants. This process can be
illustrated using Pearson correlations, which showed a sharp correlation of these two traits (soil nutrients and soil
microflora), as in Table 2.
In addition, strong positive links between soil organic matter and the other soil nutrient parameters (e.g., total
N, P and K contents) were observed in this continuous cropping system. The changes in soil organic matter based
on the application of mature compost are important. It was suggested that the application of composts to soils in
agriculture could provide long-term fertility and less off-site impacts31. However, the nutrient supply capability
of the OF treatment was efficient as compared to that of the BF. Therefore, the combination of functional PGPM
(i.e., T7) and organic fertilizer as the form of bio-organic fertilizer was the better choice to reduce the use of
chemical fertilizers and promote tomato yield and quality. After fermentation in compost, T7 utilized the nutri-
ents provided by the organic material and established themselves in it, which increased the viability of the added
T7 and made these strains more competitive in the microbial community in rhizosphere5. This may also be the
reason why the BF treatment was more effective in maintaining tomato yield compared with T7 alone (SS).
In conclusion, the results of the present work imply that the application of this Trichoderma-enriched
bio-organic fertilizer rather than applying the PGPM inoculant alone could help to avoid the overuse of expen-
sive chemical fertilizers to a significant extent without compromising the yield (at least in tomatoes). More
importantly, the use of Trichoderma-enriched bio-organic fertilizer with lower fertilizer application leads to the
enhancement of fruit quality and the improvement of soil fertility and microbial environment. There, bio-organic
fertilizers like this could be employed to supplement to the reduced rate of chemical fertilizers, and should be
further evaluated as an important component of the integrated nutrient management regime.

Materials and Methods

Microbial strain. T. harzianum strain T7 was maintained in our lab which was originally isolated from soil. It
was cultured on potato dextrose agar (PDA) at 28 °C and stored at 4 °C on slants. The strain purity was confirmed
by PCR and sequencing to exclude the contamination from bacteria and other fungi before use.

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TN TP TK OM AN NN AP AK Bac Fun Act Tri Yield Vc TSS NO3-

TN 1
TP 0.188 1
TK 0.115 0.967** 1
**
OM 0.821 0.463 0.309 1
AN −0.496 −0.233 −0.216 −0.352 1
NN 0.836** 0.590* 0.533* 0.817** 0.305 1
AP 0.920** 0150 0.091 0.716** −0.447 0.795** 1
AK 0.825** 0.483 0.457 0.762** −0.367 0.913** 0.847** 1
Bac 0.777** 0.478 0.358 0.772** −0.683** 0.714** 0.712** 0.677** 1
Fun 0.516* 0.247 0.236 0.398 −0.733** 0.480 0.504* 0.423 0.647** 1
Act 0.282 0.569* 0.450 0.551* −0.202 0.522* 0.221 0.432 0.556* 0.401 1
Tri 0.198 0.327 0.334 0.138 −0.616* 0.269 0.205 0.289 0.370 0.660** 0.568* 1
Yield −0.069 −0.412 −0.423 −0.062 −0.001 −0.234 0.092 0.007 −0.282 −0.103 −0.438 −0.108 1
Vc 0.534 −0.201 −0.327 0.525* −0.042 0.350 0.462 0.201 0.430 0.280 0.392 −0.031 −0.304 1
TSS 0.591* 0.669** 0.634** 0.699** −0.591* 0.687** 0.537* 0.657** 0.879** 0.621* 0.661** 0.402 −0.433 0.281 1
NO3- −0.538* 0.214 0.360 −0.543* 0.545* −0.173 −0.388 −0.144 −0.582* −0.373 −0.211 −0.062 −0.007 −0.667** −0.356 1

Table 2. Pearson correlations of soil nutrients and microbial abundance with tomato yield and quality.
Abbreviations: TN, Soil total N; TP, Soil total P; TK, Soil total K; OM, Soil organic matter; AN, Soil
ammonium-N; NN, Soil nitrate-N AP, Soil available P; AK, Soil available K; Bac, Soil bacteria population; Fun,
Soil fungi population; Act, Soil actinomycetes population; Tri, Soil Trichoderma population; Yield, Yield of
tomato; Vc, Vitamin C content of tomato fruit; TSS, Total soluble sugars content of tomato fruit; NO3-, NO3-
content of tomato fruit. Correlations significant at the *P < 0.05 level and **P < 0.01 level (2-tailed).

Bio-organic fertilizer preparation. Spore suspensions of T7 were prepared by flooding PDA cultures of
7-day-old T7 with sterile water; the suspensions were then filtered through a double layer of sterile cheesecloth
and adjusted to 106 conidiospores per mL based on the hemocytometer counts. The bio-organic fertilizer used
in this study was obtained by aerobically fermenting a mixed organic fertilizer with T7 spore suspension (100:1,
w/v) for 7 days at < 50 °C. The mixed organic fertilizer was prepared from mature compost of pig manure, which
contained 42.5% organic matter, 3.5% N, 2.1% P2O5, 1.2% K2O and 28.4% H2O. The final bio-organic fertilizer
was stored at 4 °C and was used in the experiments only if the population of the T7 remained at the level of 106
cfu per gram of dry matter.

Field trials design. Field trials were conducted twice at a vegetable testing site in army horse ranch 6 team
(located at longitude 106°27’ E and latitude 38°47’ N), Yinchuan, Ningxia Province, China. No specific permits
were required for the described field studies. The location is not privately owned or protected, and the field studies
did not involve endangered or protected species. The soil characteristics were pH 6.1, organic matter 27.6 g kg−1,
ammonium-N 21.9 mg kg−1, nitrate-N 22.7 mg kg−1, available P 131.3 mg kg−1 and available K 218.7 mg kg−1. The
above data were collected by measuring five soil samples from the field site before planting. The first crop season
extended from August to November; the second crop season lasted from March to June. The field treatments
were designed as (1) CF: 100% chemical fertilizer; (2) BF: 75% chemical fertilizer + bio-organic fertilizer; and
(3) OF: 75% chemical fertilizer + organic fertilizer. The 100% chemical fertilizer contained 600 kg ha−1 YaraMila
compound fertilizer (N: P: K = 15: 15: 15) and 300 kg ha−1 YaraLiva-Ca(NO3)2 fertilizer (N ≥ 15%, split into
3 × 100 kg, YARA international ASA, Oslo, Norway) and was applied in plots consistent with the local farmers’
fertilization practice. The 75% chemical fertilizer used in this study was based on the 100% fertilizer because a
75% rate was reported to be a threshold below which the PGPM-fertilizer interaction could not produce consist-
ent nutrient uptake comparable to the non-inoculated full fertilizer rates1,11. The application rate of bio-organic
fertilizer or organic fertilizer was 1800 kg ha−1. Each treatment with 5 plots was randomly arranged in the selected
fields, and each plot (6.0 m × 1.6 m) contained 60 tomato seedlings. Conventional practices like watering (every
ten days), scarifying and disinsection were equally given when needed.

Pot experiments design. To investigate the effect of T. harzianum T7 inoculation alone (without organic
substance) on tomato growth, yield and fruit quality and on the soil environment, another treatment (SS: 75%
chemical fertilizer + spore suspension) was added to the pot experiments, and further experiments were con-
ducted using the same treatments applied in the field trials. At planting, seedlings with CF treatment (100% chem-
ical fertilizer) received 5.33 g plant−1 YaraMila compound fertilizer. They were then fertigated with 2.67 g plant−1
YaraLiva-Ca(NO3)2 between four and five weeks after planting. The reduced fertilization treatments (BF, OF and
SS) received 75% of the chemical fertilizer and 50 g plant−1 bio-organic fertilizer, 50 g plant−1 organic fertilizer or
5 mL plant−1 Trichoderma spore suspension (106 cfu mL−1). Each pot (30 × 30 × 35 cm3) contained 10 kg soil and 2
seedlings. The soil used in this work was a loam (pH 7.3) with an organic matter content of 19.2 g kg−1. The available
P and K, ammonium-N and nitrate-N contents were 99.2, 150.5, 29.3 and 0.8 mg kg−1, respectively. Six pots were
used for each treatment, and they were arranged randomly in the greenhouse, with a maintained temperature of
20–30 °C. The pot experiments were continuously carried out for four times with repeated fertilization and planting.

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Estimation of tomato yield and fruit quality parameters. Destructive harvest was conducted on the
100th day after planting in both field trials and pot experiments, and data regarding the tomato yields were col-
lected. Ripe fruits were harvested 3 times manually from 60 plants for each plot in the field trials (10 plants for pot
experiments), and 3 of the fruits were selected to form a replicate for each treatment.
The contents of Vc and nitrate (expressed in NO3-) in the tomato fruit were analyzed with an Agilent 1200
semi-preparative HPLC (Agilent Technologies, Santa Clara, Cal., USA). For the Vc content, 5 g of fruit samples
were homogenized in 50 mL of ice-cold extraction buffer (5 g L−1 oxalic acid) for 15 min and then transferred to a
100 mL volumetric flask diluted with the extraction buffer. The homogenate was filtered through a 0.45 μm filter
membrane. All of the procedures were performed at 4 °C. The mobile phase was composed of 0.05 M KH2PO4
solution and methanol at a ratio of 95:5 (v/v) and used at a flow rate of 1.0 mL min−1; the eluate was detected at
265 nm at room temperature. The standard curve of Vc was developed with ascorbic acid (Sigma, USA) from
5 mg L−1 to 100 mg L−1. To evaluate the nitrate content in the tomato fruit, 50 g of fresh fruit was homogenized in
50 mL of deionized water. Five grams of the homogenate was added to 50 mL of hot water (70–80 °C) and used
for ultrasonication for 20 min. After cooling, 5 mL of the above extract was diluted to 25 mL with deionized water
and filtered through a 0.45 μm filter membrane. The eluent was 0.03 M KH2PO4-H3PO4 buffer (pH = 3.3) at a
1 mL min−1 flow at room temperature at 210 nm. Standard control was set up from 0.1 mg L−1 to 10 mg L−1 with
KNO3. The TSS content was measured using an anthrone colorimetric method at 620 nm as described by Allen32.

Soil sampling and nutrient content analysis. A composite soil sample (with three replicates) was col-
lected from the pot soil which was mixed thoroughly manually before splitting into pots and was referred to as
the 0th soil sample. At every harvest, soil samples were collected from the harvested plant roots of each treatment
as described by Hervás et al.33 and referred to as the 1st, 2nd, 3rd and 4th soil samples. The tomato plants were
carefully removed from the pots and gently shaken to detach the soil. Soils still adhering to the roots were defined
as the rhizosphere soil samples. The soil samples for the field trials were prepared at harvest as described above
for the pot experiment sampling.
The total N content and organic matter of soil samples were analyzed using a Vario EL elemental analyzer
(Elementar Analysensysteme GmbH, Hanau, Germany). The total P and total K were analyzed using inductively
coupled plasma-atomic emission spectrometer (Agilent 710 ICP-OES, Agilent Technologies, Santa Clara, Cal.,
USA). The soil available nutrients, including soil available P and available K, were determined following Shen
et al.34, and the soil available N (ammonium-N and nitrate-N) was analyzed using a continuous-flow analyzer
(AutoAnalyzer 3, Bran + Luebbe GmbH, Germany).

Soil cultivable microbial count. The populations of soil cultivable bacteria, fungi, actinomycetes and
Trichoderma were detected using the standard 10-fold dilution plating method. Specifically, Beef extract medium
was used for the characterization of total bacteria population, Martin medium for fungi, Gause NO.1 medium for
actinomycetes35 and a modified Trichoderma selective medium [1 L contained water, 800 mL; agar, 20 g; glucose,
3 g; NH4NO3, 1 g; KH2PO4, 0.9 g; MgSO4·7H2O 0.2 g; KCl, 0.15 g; rose Bengal, 0.15 g; chloramphenicol, 0.25 g;
streptomycin, 0.05 g; quintozene (PCNB), 0.15 g; Captan, 0.1 g; propamocarb (772 g of active ingredient per liter),
1.2 mL; and Triton X-100, 1 mL] based on TSM medium36 for Trichoderma population. The data were expressed
as the number of cfu per gram dry soil.

Statistical analysis. The results were expressed as the means ± standard deviations and were calculated and
statistically examined using an analysis of variance and a Duncan’s multiple range test. Statistical significance was
considered at P < 0.05, unless otherwise stated. Most tests were conducted using SPSS version 13.0 (SPSS, Inc.,
Chicago, IL, USA). The Pearson correlation coefficient of pairwise correlation analysis was performed using SPSS
to relate the tomato yield, quality and soil chemical and biological properties.

Received: 27 March 2019; Accepted: 11 December 2019;

Published: xx xx xxxx

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Acknowledgements
This research was funded by the key research and development projects of Ningxia Hui Autonomous Region
(Grant No. 2018BBF02009 and 2018BBF03001), Ningxia University first-class discipline construction project
(Grant No. NXYLXK2017B03), and the Natural Science Foundation of China (Grant No. 31801903). We are
grateful to the Institutes of Ningxia protected horticulture engineering technology research center and Ningxia
protected horticulture (Ningxia University) technology innovation center for supporting.

Author contributions
L.Y. and J.L. conceived the study. L.Y. designed the experiments. L.Y. and X.Z. conducted the experiments and
the data analysis. K.C., E.B. and Z.Z. wrote and revised the manuscript. All authors read and approved the final
manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-019-56954-2.
Correspondence and requests for materials should be addressed to L.Y. or Z.Z.
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