Research Article

Horticultural Science and Technology. August 2021. 431-445
https://doi.org/10.7235/HORT.20210039

ABSTRACT


MAIN

  • Introduction

  • Materials and Methods

  •   Rhizosphere Soil Collection

  •   Culture Media

  •   Soil Microbial Isolation and Purification

  •   Physiological and Biochemical Identification

  •   DNA Extraction and Illumina Sequencing

  •   Sequence Alignment and Cluster Analysis

  • Results

  •   Soil Micro-Ecology

  •   Colony Characteristics and Microscopic Morphology of Bacteria, Actinomycetes, and Fungi in the Soil of Continuously Cropped Pepper

  •   Physiological and Biochemical Properties of Bacteria and Actinomycetes in the Soil of Continuously Cropped Pepper

  •   Sequencing and Cluster Analysis of Bacteria, Actinomycetes, and Fungi in the Soil of Continuously Cropped Pepper

  • Discussion

Introduction

Pepper blight caused by Phytophthora capsici devastates pepper production and can spread quickly in a short time (Kim et al., 2002; Zhang et al., 2008; Barchenger et al., 2018). Since its isolation from pepper for the first time in 1918 in New Mexico, United States (Leonian, 1922), nearly 80 pathogenic species have been found residing in soil in various states such as mycelium, sporangium, chlamydospores, and spores (Judelson and Blanco, 2005). Therefore, P. capsici not only has the ability to resist rhizosphere stresses, but can also easily activate to infect pepper. New sporangium and zoospores on pepper spread in the soil by rain or through irrigation water, which could lead to a large infestation in the area at the seedling stage. It may also spread through the air, infecting the plant stem, leaves, flowers, and fruits (Silvar et al., 2006). Recently, large production losses caused by pepper blight have been documented in many countries with particularly devastating effects in the United States, Brazil, India, Mexico, Russia, South Korea, Japan, Bulgaria, France, Italy, Argentina, and Pakistan (Hao et al., 2007; Barchenger et al., 2018; Nawaz et al., 2018; Reyes et al., 2019; Reyes et al., 2019). In China, pepper blight was first discovered in Jiangsu province in the 1950s; more than 10 provinces are reporting serious pepper blight infections, especially in the north (Yao et al., 2016). It is the most severe pathogen in the agricultural sector and causes blackening of the vegetable roots with subsequent wilting and necrosis in the earlier stage and plant death in the later stages, resulting in a >20% loss in harvest yield.

Continuous cropping is common in agricultural production and can lead to a series of issues such as degradation of soil quality, aggravation of diseases and pests, and reductions in crop yield (He et al., 2008; Wu et al., 2015). It is the main reason for the spread of soil-borne diseases due to nutrient imbalances, decreases in enzyme activity, and physical/chemical and microflora disturbances (Li et al., 2016). Soil microbes play key roles in soil micro-ecology such as organic matter decomposition, nutrient transport, purification of environmental pollutants, and mediation of greenhouse gases (Kennedy and Smith, 1995; Berendsen et al., 2012; Zhao et al., 2015). Soil microbial activity and respiration intensity are closely related to soil catalase activity, which is an important indicator of the soil micro-ecological environment. Li et al. (2016) reported that the relative abundance of dominant bacteria increases considerably with long-term consecutive monoculture in the rhizosphere of black pepper, while an increasing trend has been observed in the abundance of pathogenic Fusarium fungi paired with a decrease in Pseudomonas and Bacillus bacteria. Similarly, long-term continuous cropping causes a significant decrease in soil pH and organic matter enzymatic activities and results in bacterial decline in black pepper soil (Xiong et al., 2015). This promotes soil microbial imbalances and reduces the number of beneficial microbes while increasing pathogenic microorganisms, leading to a higher susceptibility of plants to various soil-borne diseases. The plant’s ability to counter soil-borne disease and the activity of soil enzymes are closely related to the composition of microbes present in the rhizosphere (Magnuson and Crawford, 1992; Simoes et al., 1997). Therefore, improving the soil environment is critical for countering the detrimental effects of continuous cropping. However, there are few studies on the effect of continuous pepper cropping on the microbial community composition in the rhizosphere over time.

The main methods of prevention and control of pepper blight are the use of resistant pepper varieties and chemical agents. However, the lack of resistant varieties and the problems of using chemical agents limit the prevention and control of pepper blight(Hartman and Wang, 1992; Hai et al., 2013; Barchenger et al., 2018). Therefore, biological control has become an attractive option and has been studied by many researchers, in which screening for bacteria that have the ability to control pepper blight is a key process (Sid et al., 2003; Wang et al., 2019). Many microorganisms with the ability to control pepper blight have been isolated from plants and rhizosphere soil including Bacillus sp strains, Trichoderma harzianum strains, and Actinomycetes sp strains (Anitha et al., 2003; Tran et al., 2008). Twelve isolates have been obtained from the pepper rhizosphere, and one strain, named PS119, has a remarkable ability to suppress the growth of P. capsici (Rajkumar et al., 2005). Aspergillus sp strains are widely distributed saprophytic fungi that have been used industrially in processes such as alcohol fermentation, biological engineering and transformation, and genetic research to produce antibiotics, organic acids, and enzymes (Berka et al., 1992; Roehr et al., 1992; Aybeke et al., 2014; Park et al., 2017). However, there are limited reports of using Aspergillus sp strains to control pepper blight.

To determine the microbial community and diversity in continuously cropped pepper soil, we selected soil that had been subjected to continuous cropping for 3, 6, and 9 years and identified the number of bacteria, fungi, and actinomycetes in the rhizosphere by dilution plating. The morphological, physiological, and biochemical characteristics as well as 16S ribosomal DNA (rDNA) or rDNA internal transcribed spacer (ITS) sequences were examined to study the soil microbial ecology. These findings provide the foundation for optimising the soil micro-environment to control pepper blight.

Materials and Methods

Rhizosphere Soil Collection

The experimental soil samples were collected from different long-term pepper greenhouse fields that were consecutively cropped for 3, 6, and 9 years with the same agronomic management and fertilisation regime in Beizhen city, Liaoning province of China. Soil that was less than 5 mm from the root surface was gathered and defined as the rhizosphere. The rhizosphere soil of healthy and diseased pepper plants was collected in the same greenhouse, and six treatments were tested. Three greenhouses were randomly selected for each pepper planting year. The five-point sampling method was used in each greenhouse (Xin et al., 2006), and for each pepper field, five healthy and P. capsici-infected pepper plants were randomly collected with intact roots and surrounding soil by shaking (Dong et al., 2013). Each treatment with three replicates included 15 sample sites and 75 rhizosphere soil of pepper. All soil samples were rapidly frozen with liquid nitrogen and stored at -80°C for DNA extraction.

Culture Media

Luria-Bertani (LB) media was used for bacteria isolation and culture, Potato-Martin substratum was used for fungi isolation and culture, and Gao No. I media was used for actinomycetes isolation and culture. The compositions of the media are shown in Suppl. Table 1s (Fang, 1998; Dong and Cai, 2001).

Soil Microbial Isolation and Purification

Dilution plating was used to isolate bacteria, fungi, and actinomycetes from different long-term consecutively cropped soils. The ratio of microbes between healthy and diseased soil was calculated as follows: R (%) = (N1-N2)/N2 × 100% where N1 represents the amount of bacteria, fungi, and actinomycetes in diseased soil and N2 is the amount in healthy soil.

Physiological and Biochemical Identification

The physiological and biochemical index, catalase reaction, methyl red (MR) reaction, V-P reaction, starch hydrolysis reaction, cellulose decomposition, nitrate reduction, nitrite reduction, H2S, lipase, tryptophan deaminase enzyme, phenylalanine deaminase, citrate utilization, and salt tolerance were examined for physiological and biochemical identification of bacteria and actinomycetes.

DNA Extraction and Illumina Sequencing

The DNA extraction kits for bacteria and fungi (Tiangen Biotech, China) were used to extract bacterial and fungal DNA, which was examined by 1.2% agarose gel electrophoresis. The OD260 and OD280 values were also measured, and OD260/280 was calculated to determine the concentration and purity of DNA. We used 2× Taq PCR Master Mix kits (Tiangen Biotech, China) for PCR amplification, after which we sequenced the amplification products. Each reaction contained 12.5 µL master mix, 1.0 µL DNA, 0.5 µL of each primer, and 14 µL sterile distilled water. The PCR cycle was as follows: 94°C denaturation for 3 min, 94°C denaturation for 30 sec, 55°C annealing for 30 sec, and 72°C extension for 1 min for 30 cycles followed by 72°C extension for 5 min. Bacteria and actinomycetes 16S rDNA PCR primers were 5'-AGAGTTTGATCCTGGCTCAG-3' (forward) and 5'-GGTTACCTTGTTACGACTT-3' (reverse). Fungal rDNA-ITS PCR primers were 5'-TCCTCCGCTTATTGATATGC-3' (forward) and 5'-GGAAGTAAAAGTCGTAACAAGG-3' (reverse).

Sequence Alignment and Cluster Analysis

The obtained sequences were blasted in the NCBI database. Clustalx1.83 was used to construct an alignment, and MEGA 4.0 was used to infer phylogenetic trees. Bacteria and actinomycetes were identified by colony and cell morphology, gram staining, physiological and biochemical tests, and 16S DNA sequence analyses. Fungi were identified by colony and cell morphology and rDNA-ITS sequence analysis.

Results

Soil Micro-Ecology

Bacteria including actinomycetes and fungi in the soil drive the soil ecosystem’s biochemical cycle (Cao et al., 2011). To understand the effects of continuous cropping of pepper on the rhizosphere soil ecosystem over time, we studied the number of cultivatable bacteria, actinomycetes, and fungi in farms after 3, 6, and 9 years (3a, 6a, and 9a, respectively) of continuous cropping (Table 1). The number of bacteria in the rhizosphere of healthy plants at 3a, 6a, and 9a was significantly higher than in diseased soil and showed a peak at 6a. We did not observe a significant difference in bacteria quantity in healthy plants at 3a, 6a, and 9a or diseased plants at 3a and 9a. However, the number of bacteria in diseased plants at 6a was significantly elevated. In addition, a comparison between the number of rhizosphere bacteria in healthy and diseased plants in the same year showed consistently lower bacteria counts at all time points. We observed the largest decrease in diseased plants at 9a followed by the 3a and 6a time points. Similarly, the number of rhizosphere actinomycetes in healthy plants at 3a, 6a, and 9a was slightly higher than in diseased soil but showed an inverse temporal trend compared with bacteria. Furthermore, the number of rhizosphere actinomycetes was significantly increased at 9a in healthy plants, while we did not detect any significant differences in diseased plants. Actinomycete counts at 3a, 6a, and 9a in diseased plants were 7.67%, 5.42%, and 12.35% lower, respectively, compared with healthy plants. Compared with rhizosphere bacteria, the decrease in actinomycetes in diseased plants was relatively mild. Conversely, the number of fungi in the rhizosphere of diseased plants was significantly higher than in healthy plants with a peak at 6a. We observed increases of 37.82%, 19.75%, and 17.29% at 3a, 6a, and 9a, respectively. In healthy plants, the number of fungi was significantly elevated at 6a compared with 3a, but no difference was found between 6a and 9a. However, in diseased plants, the fungi count continued to rise significantly at 9a.

Table 1.

Quantity of soil microbes in the continuously cropped soil of healthy and diseased pepper plants

Consecutive years Average number of Bacteria
(108 CFU/mL)
Average number of Actinomycetes
(106 CFU/mL)
Average number of Fungi
(104 CFU/mL)
Healthy plants Diseased plants Healthy plants Diseased plants Healthy plants Diseased plants
3 a 3.44 ± 0.36 c 0.64 ± 0.13 b 3.13 ± 0.33 b 2.89 ± 0.27 a 8.09 ± 0.37 b 11.15 ± 0.45 c
R (%) 537.50 108.31 72.56
6 a 5.55 ± 0.31 a 1.19 ± 0.16 a 2.95 ± 0.46 b 2.79 ± 0.23 a 12.16 ± 0.50 a 19. 75 ± 0.82 a
R (%) 466.39 105.74 61.57
9 a 4.49 ± 0.33 b 0.48 ± 0.10 b 4.29 ± 0.33 a 3.76 ± 1.25 a 11.89 ± 0.40 a 17.29 ± 0.42 b
R (%) 935.42 114.10 68.77

R: ratio of healthy plants to diseased plants.

The results are given as the mean ± SE.

Different letters indicate a significant difference between treatments by Duncan’s post-hoc test at p < 0.05 (n = 3).

The bacteria/fungi ratio reflects the structure and nutritional function of the soil food chain. We found significantly higher bacteria/fungi ratio at 3a, 6a, and 9a in healthy plants compared with diseased plants (Table 2), which decreased by 761.67%, 766.67%, and 1266.67%, respectively. The decrease was highest at 9a, indicating that the growth rate of bacteria was significantly higher than that of fungi. In both healthy and diseased soil, bacteria dominated in absolute numbers followed by actinomycetes and fungi (Tables 1 and 2). Taken together, the number of bacteria and actinomycetes in the rhizosphere of healthy plants was higher than in diseased plants, while the number of fungi was lower. Infection by P. capsici had the greatest impact on the number of soil bacteria and the least impact on fungi and actinomycetes. Time point 6a appeared to be the pivotal point at which the microbial composition changed significantly.

Table 2.

Proportion of bacteria, fungi, and actinomycetes in the continuously cropped soil of healthy and diseased pepper plants

Consecutive years B/F (104) A/F (102) B/A (102)
Healthy plants Diseased plants Healthy plants Diseased plants Healthy plants Diseased plants
3 a 0.43 ± 0.05 a 0.06 ± 0.01 a 0.39 ± 0.06 b 0.26 ± 0.03 a 0.92 ± 0.15 a 4.52 ± 0.53 b
R (%) 716.67 150.00 20.36
6 a 0.46 ± 0.02 a 0.06 ± 0.01 a 0.24 ± 0.05 a 0.14 ± 0.01 b 0.53 ± 0.10 b 2.34 ± 0.47 c
R (%) 766.67 171.43 22.65
9 a 0.38 ± 0.04 a 0.03 ± 0.01 b 0.36 ± 0.02 b 0.22 ± 0.07 ab 0.96 ± 0.16 a 7.90 ± 1.33 a
R (%) 1266.67 163.64 12.15

R: ratio of healthy plants to diseased plants.

B/F : ratio of bacteria to fungi; A/F: ratio of actinomycetes to fungi; B/A: ratio of bacteria to actinomycetes.

The results are given as the mean ± SE.

Different letters indicate a significant difference between treatments by Duncan’s post-hoc test at p < 0.05 (n = 3).

Colony Characteristics and Microscopic Morphology of Bacteria, Actinomycetes, and Fungi in the Soil of Continuously Cropped Pepper

Bacteria were isolated from soil by dilution plating, and the morphology of the main colony is shown in Table 3. We performed a Gram stain and observed the microscopic morphology using a microscope at ×100 magnification (Table 4).

The isolated actinomycetes with dry, wrinkled, and concentric growth characteristics were selected, inoculated on actinomycete culture medium, and cultured at 28°C for 5–7 d. The colony morphology was then noted (Table 3). Glass slides were inserted into the actinomycete culture medium to examine the spore morphology after 7 d (Table 4). Fungi were isolated from soil, inoculated on PDA medium, and incubated at 25°C for 5–7 d. The colony morphology was then observed (Table 3), and the mycelia were isolated onto plates and examined using an optical microscope (Table 4).

Table 3.

Morphology of bacterial, actinomycete, and fungal colonies in the soil of continuously cropped pepper

Microbial No. Colony morphology
Bacterial B15 Irregular shape, beige, not raised, not shiny, dry, opaque, irregular edges,
wrinkled,easy to pick, combined with the medium does not close,
some colour on both sides, no pigment production
B16 Round, white, raised, moist, shiny, opaque, irregular edges, wrinkled, sticky,
easy to pick, combined with the medium does not close,
some colour on both sides, no pigment production
B22 Round, white, convex, moist, shiny, neat edge, opaque, easy pick,
combined with the medium does not close, some colour on both sides,
no pigment production
B23 Round, yellowish, round, neat edges, raised slightly, moist and shiny,
opaque, easy to pick, combined with the medium does not close,
some colour on both sides, no pigment production
B32 Round, yellowish, neat edges, smooth, shiny, waxy, no pigment production
B33 Small colony, round, white, neat edge, not moist, not shiny, opaque, not raised,
easy to pick, some colour on both sides, no pigment production
B40 Large colonies, round, white, wrinkled, irregular edges, not moist, not shiny,
opaque, not raised, easy to pick, some colour on both sides,
no pigment production
B41 Round, yellow, opaque, smooth, neat edge, raised, no pigment production
B43 Round, white, opaque, moist, shiny, not raised, irregular edges, easy to pick
B44 Round, white, opaque, not moist, not shiny, wrinkled, not raised, irregular edges,
easily provoked, some colour on both sides, no pigment production
B45 Small colony, round, orange, neat edges, moist and shiny, opaque, easy to pick,
some colour on both sides, no pigment production
B47 White, raised, neat edge, moist, shiny, translucent, viscous, convex colonies,
easy to pick, some colour on both sides, no pigment production
B48 Round, white, opaque, raised slightly, no pigment produced
B51 Gray, jagged, transparent, easy to pick, some colour on both sides,
no pigment production
B52 Round, white, not shiny, opaque, hard to pick, no pigment production
Actinomycete A3 Round, neat edges, concentric circles, inside of colony is reddish brown while edge of
colony is white, hard and difficult to pick, opaque, grey spore powder, dry,
same colour on both sides, no pigment production
A4 Irregular shape, white, jagged, dry, not shiny, opaque, same colour on both sides,
brown pigment production
A6 Faint yellow colony, pink and white aerial mycelium, opaque, closely combined with the medium,
no pigment production
A7 Inside of the colony is creamy yellow, while the edge of it is white, irregular shape, hard, dry,
opaque, same colour on both sides, no pigment production
A9 Aerial mycelium is white, substrate mycelium is dark blue, brown pigment production
A11 Colony is white, irregular shape, opaque, dry, same colour on both sides,
no pigment production
A12 Round, creamy yellow, hard, difficult to pick, opaque, spore colour is white to light pink,
same colour on both sides, no pigment production
A13 Round, brown, neat edge, hard and thick, difficult to pick, surface spores white to pale yellow,
dry, colonies on the back of the same colour,
no pigment production
A14 Colony is grey, hard and thick, opaque and difficult to pick, reverse side is light brown,
no pigment production
A20 Center of colony is milky white and edge of colony is white, jagged, wrinkled, not shiny,
opaque, same colour on both sides, no pigment production
A26 Round, white, hard thick and difficult to spick, dry, same colour on the both sides,
no pigment production
A53 Irregular shape, white, jagged, dry, not shiny, opaque, same colour on the both sides,
no pigment production
A55 Small colony, center of colony is pink and edge of colony is red, neat edge,
hard and difficult to pick, dry, white spore, opaque,
reverse side of the colony is red
A60 Round colony, white, irregular edges, dry, opaque, not shiny, hard to spick,
same colour on the both sides, no pigment production
A61 White colony, hard and difficult to spick, opaque, hard and thick, white spore,
dry, same colour on both sides, no pigment production
A62 Small colony, round, white, neat edge, raised, opaque, not moist, not shiny,
same colour on both sides, no pigment production
A63 Round, grey, neat edges, dense, hard and difficult to pick, opaque, white spore,
dry, reverse side of colony is black, brown pigment secretion production
Fungi F17 The colony is yellow in the beginning and then the colour changes to yellow-green
F18 The colony is round, white, and fluffy, lots of green conidium can be produced,
the resulting colour of the centre of the colony is green,
but the outside colony is also white
F21 The colony is white, mycelia are few and scattered, combined with the media closely,
non-pigment production
F27 The colony is green and fluffy, the colour of the centre of the colony is green,
but outside of the colony is milky white
F19 The colony is white to yellowish, sometimes it is blue, aerial hyphae like light fleece
F66 The colony is pink or pale purple, aerial mycelium colour changes from white and
pink to light pale purple, aerial mycelium like light fleece.
Table 4.

Microscopic morphology of bacteria, actinomycetes, and fungi in the soil of
continuously cropped pepper

Microbial No. Microscopic morphology
Bacterial B15 G+, rod-shaped bacteria with spores
B16 G+, oval to rod-shaped bacteria with spores, sporangium enlargement
B22 G+, rod-shaped bacteria, oval spore, sporangium enlargement
B23 G, rod-shaped bacteria, non-spore
B32 G, short rod bacteria without spore
B33 G, rod-shaped bacteria without spore
B40 G, short rod bacteria without spore
B41 G+, spherical bacteria with spore
B43 G+, rod-shaped bacteria with spore
B44 G+, rod-shaped bacteria with spore
B45 G+, rod-shaped bacteria with spore
B47 G, rod-shaped bacteria without spore
B48 G+, spherical bacteria without spore
B51 G+, rod-shaped bacteria with spores
B52 G+, spherical bacteria without spore
Actinomycete A3 Spore is bent, spherical, or ovoid.
A4 Sporothrix is slender and branching, the spore is oval.
A6 Sporothrix is spiral; the spore is cylindrical
A7 Sporothrix is bent; spore is oblong
A9 Sporothrix is bent; spore is oval
A11 Sporothrix is bent; spore is oval
A12 Sporothrix is bent; spore is oval
A13 Sporothrix is dense and spiral; spore is oval
A14 Sporothrix is curved or spiral; spore is oval
A20 Sporothrix is long with little spirals; spore is spherical to oval
A26 Sporothrix is dense with big spiral; spore is oval
A53 Sporothrix is long and curved
A55 Sporothrix is long and curved
A60 Sporothrix is bent
A61 Sporothrix is relatively straight; spore is cylindrical
A62 Sporothrix is bent
A63 Sporothrix is long and straight
A3 Spores is spherical and oval with smooth surface
Fungi F17 There are many complex branches; part of them produces many long and rough conidiophores,
flask-shaped tip produces top-sac or nearly spherical surface produces many small stems (usually double),
a small terrier on the generator the spherical surface roughness string conidia
F18 1 or 2 times broom branches; conidia are nearly spherical to oval
F21 Mycelium is filamentous, slender, and without diaphragm; most of them are at a right angle or
an acute angle with constriction; sporangiophore is colourless, filamentous;
sporangia are ovoid or pear-shaped
F27 There are many sterigma; conidia are oval
F19 Microconidia are oval or kidney-shaped; macroconidium is sickle-shaped, both ends are blunt,
end spores are bent slightly, and most of macroconidia have three diaphragms.
F66 Microconidia are oval or kidney-shaped; macroconidium is sickle-shaped; ends of them are sharpened

Physiological and Biochemical Properties of Bacteria and Actinomycetes in the Soil of Continuously Cropped Pepper

A set of 15 bacteria and 17 actinomycetes were selected for physiological and biochemical identification and examined by physiological and biochemical indexes, hydrogen peroxide production, MR, V-P, starch and cellulose hydrolysate content, and gelatine liquefaction ability (Table 5).

Table 5.

Physiological and biochemical properties of bacteria and actinomycetes in the soil of continuously cropped pepper

No. Results Physiological and biochemical index
Peroxide MR V-P Starch
Hydrolysis
Cellulose
Hydrolysis
Gelatin
Liquefaction
Nitrate
Reduction
H2S Lipase NaCl
Growth
Citric acid
Salt use
Phenylalanine
Deaminase
10% 15%
B15 Paenibacillus sp + +- + + + + + +
B16 Paenibacillus
polymyxa
+ -+ + + + +
B22 Bacillus
amyloliquefaciens
+ + + + + + +
B23 Ochrobactrum sp + -+ + + + + + +
B32 Marseille
genus
+ -+ + + +
B33 Flavobacterium sp + +- + + + + + +
B40 Flavobacterium sp -+ + + + +
B41 Agrococcus sp + + + + +
B43 Bacillus sp + + + + + + + +
B44 Bacillus
amyloliquefaciens
+ + + + + + +
B45 Brachybacterium sp
Mn32
-+ + + +
B47 Agrobacterium sp + + +
B48 Staphylococcus
warneri
+ -+ + + + + + + +
B51 Bacillus
cereus
+ + + + +
B52 Staphylococcus
pasteuri
+ -+ -+ + + + +
A3 Streptomyces
xanthophaeus
+ + +
A4 S. oeidiscabies + + + + + + +
A6 S. microflavus + + + + + +
A7 S. fimicarius + + + + + + +
A9 S. coelicoflavus var
yongchunensis Liu
+ + + + + + +
A11 S. fradiae + + + + + + + + +
A12 S. virginiae + + + + + + +
A13 S. alanosinicus + + + + + + +
A14 S. sp + + + + + + + +
A20 S. antibioticus + + + + + + + + +
A26 S. lavendulae + + + + + + +
A53 S. oeidiscabies + + + +
A55 S. sp + + + + +
A60 S. sp + + + + + +
A61 Amycolatopsis sp + + + + +
A62 S. avermitilis + + + + +
A63 S. sp + + + + + +

Sequencing and Cluster Analysis of Bacteria, Actinomycetes, and Fungi in the Soil of Continuously Cropped Pepper

Identification was performed according to the common bacteria system identification manual. Bacterial, actinomycete, and fungal DNA was extracted and detected by 1.2% agarose gel electrophoresis (Figs. 1A, 2A and 3A). We calculated an OD260/280 value of 1.8 to 2.0 and performed 16S rDNA PCR. The results indicated that the size of the bacterial DNA amplification sequences B22 and B15 were about 1000 bp, and the remaining sequences were 1500 bp (Fig. 1B). In comparison, actinomycete and fungi sequences were 2000 bp long (Figs. 2B and 3B). We then sequenced the PCR products and found that the nucleotide fragments of bacteria, actinomycetes, and fungi were 952–1466 bp, 948–1440 bp, and 500–750 bp, respectively (Table 6). The sequences were compared using the NCBI database, and Clustalx1.83 was used to construct an alignment. MEGA 4.0 was employed for cluster analyses of the respective bacteria and actinomycetes.

/media/sites/kshs/2021-039-04/N0130390403/images/HST_39_04_03_F1.jpg
Fig. 1.

(A) Genomic DNA extraction and (B) PCR amplification of bacteria isolated from the soil of continuously cropped pepper.

/media/sites/kshs/2021-039-04/N0130390403/images/HST_39_04_03_F2.jpg
Fig. 2.

(A) Genomic DNA extraction and (B) PCR amplification of actinomycetes isolated from the soil of continuously cropped pepper.

/media/sites/kshs/2021-039-04/N0130390403/images/HST_39_04_03_F3.jpg
Fig. 3.

(A) Genomic DNA extraction and (B) PCR amplification of fungi isolated from the soil of continuously cropped pepper.

Table 6.

Sequencing and cluster analysis of bacteria, actinomycetes, and fungi in the soil of continuously cropped pepper

Number Known sequences Max ident (%) Taxonomic position
B15 AB680894.1 Paenibacillus peoriae 99 Paenibacillus peoriae
B16 JN700208.1 Paenibacillus polymyxa 98 Paenibacillus polymyxa
B22 N998403 Bacillus amyloliquefaciens 100 Bacillus amyloliquefaciens
B23 JN256921.1 Ochrobactrum sp 99 Ochrobactrum sp
B32 JF460770.1 Massilia alkalitolerans 98 Massilia alkalitolerans
B33 HE681208.1 Flavobacterium sp 99 Flavobacterium sp
B40 JQ723710.1 HM303244 Flavobacterium sp 99 Flavobacterium sp
B41 JF135709 Agrococcus sp 99 Agrococcus sp
B43 JF496513 Bacillus thuringiensis 100 Bacillus thuringiensis
B44 JN661699.1 Bacillus amyloliquefaciens 98 Bacillus amyloliquefaciens
B45 GU377106.1 Brevundimonas sp 99 Brevundimonas sp
B47 HM151906.1 Agrobacterium sp 99 Agrobacterium sp
B48 JN208111.1 Staphylococcus warneri 98 Staphylococcus warneri
B51 JN700160 Bacillus cereus 99 Bacillus cereus
B52 FR839669 Staphylococcus pasteuri 97 Staphylococcus pasteuri
A3 DQ442560.1 Streptomyces xanthophaeus 99 Streptomyces xanthophaeus
A4 JF923433.1 Streptomyces acidiscabies 100 Streptomyces acidiscabies
A6 FN646655.1 Streptomyces microflavus 99 Streptomyces microflavus
A7 JQ342917.1 Streptomyces fimicarius 99 Streptomyces fimicarius
A9 JQ409522.1 Streptomyces coelicoflavus 99 Streptomyces coelicoflavus
A11 JQ409522.1 Streptomyces fradiae 99 Streptomyces fradiae
A12 FJ481068 Streptomyces virginiae 99 Streptomyces virginiae
A13 HQ426712 Streptomyces alanosinicus 98 Streptomyces alanosinicus
A14 JQ320495.1 Streptomyces spororaveus 99 Streptomyces spororaveus
A20 EF063450 Streptomyces antibioticus 100 Streptomyces antibioticus
A26 FJ517747 Streptomyces lavendulae 100 Streptomyces lavendulae
A53 FJ007427 Streptomyces acidiscabies 99 Streptomyces acidiscabies
A55 HE681150.1 Streptomyces sp 100 Streptomyces sp
A60 JQ234945.1 Streptomyces sp 99 Streptomyces sp
A61 NR_042040 Amycolatopsis coloradensis 99 Amycolatopsis coloradensis
A62 JN392399.1 Streptomyces avermitilis 98 Streptomyces avermitilis
A63 JQ419707.1 Streptomyces sp 98 Streptomyces sp
F17 AB369896.1 Aspergillus flavus 99 Aflatoxin
F18 JN938952.1 Penicillium expansum 99 Penicillium expansum
F21 AF242821.1 Phytophthora capsici 99 Phytophthora
F27 GU566206.1 Penicillium oxalicum 100 Penicillium oxalate
F19 JN390707.2 F. solani isolate 99 Fusarium solani
F66 GU566205.1 Fusarium oxysporum 100 Fusarium

Discussion

Bacteria play an important role in changing soil fertility and structure and promoting nutrient circulation. In this study, we showed that the number of bacteria and actinomycetes over time in the rhizosphere of healthy and P. capsici-infected pepper plants first increased followed by a decrease. Other studies have observed that the bacterial communities in the rhizosphere of Rehmannia glutinosa significantly decrease in quantity and diversity with continuous cropping, which impairs the growth of R. glutinosa and its underground tubers and reduces its usability as a medicinal ingredient (Zhang et al., 2010). In addition, Wu and Wang (2007) and Qin et al. (2015) observed similar trends in soil bacteria from continuously cropped cucumber and potato (Wu and Wang, 2007; Qin et al., 2015). However, the results of our research showed some discrepancies with these studies, possibly due to the prolonged period of continuous cropping of up to 9 years included in our study. Vegetable farmers increase the application of fertilisers and pesticides at the early stages of discovering P. capsici infection, which artificially changes the original nutrients present in the soil followed by adaptations in the number of soil microorganisms and the structure of the flora (Yang et al., 2000; Van Schoor et al., 2009; Meng et al., 2012; He et al., 2014). We showed that after infection, the pepper plant’s rhizosphere bacteria decreased significantly, whereas actinomycetes showed no significant changes and the number of fungi increased. These observed changes in bacteria and fungi were consistent with barley root rot (Li et al., 2017), tobacco bacterial wilt (Li et al., 2020), and banana wilt (Deng et al., 2011). An explanation for this phenomenon is that the number of pathogenic fungi in the soil suddenly increases after disease and directly competes with the resident bacteria, actinomycetes, and fungi in the soil for nutrition and space, which leads to a decline in the number of beneficial microorganisms and changes the soil from “bacterial type” to “fungal type.”

The stark changes in the ratio of bacteria/fungi and actinomycetes/fungi in this study all appeared in the sixth year. This is possibly due to shifts in the absorption ability and viability of the pepper roots after prolonged continuous cropping, leading to different compositions in root exudates. This may contribute to altering the composition of microorganisms in the soil and the subsequent detrimental effects. Similar observations have been reported previously. Zhu et al. (2019) studied the number of microorganisms in the rhizosphere soil of tobacco after 2, 4, and 6 years of continuous cropping and found that 4 years is the pivotal point when the number of bacteria, actinomycetes, and fungi in the soil change significantly (Zhu et al., 2019). Furthermore, Wu and Wang (2007) reported that the soil’s microbial abundance and diversity of continuously cropped cucumber after 7 years is significantly lower than after 2 years (Wu and Wang, 2007). Crop rotation is a means of improving the soil’s micro-ecological environment and increases the yield and quality of agricultural products. Dong et al. (2019) found that when garlic and maize are rotated with pepper, the soil’s microbial community structure and diversity is improved paired with a reduction in the threat of pathogens (Dong et al., 2019). Zhang et al. (2015) found that cucumber and leafy vegetable rotation increases the number of soil microorganisms and reduces soil salinity (Zhang et al., 2015). Therefore, the data of our study suggest that when peppers are continuously planted for 5–6 years, it would be beneficial to rotate them with different crops such as garlic or leafy vegetables (Dong et al., 2019), thus improving the soil’s nutrients and increasing enzyme activity and microorganism diversity. These factors would ultimately promote the healthy and sustainable agriculture of peppers.

Seven of the 15 strains of bacteria obtained in this study belonged to the genus Bacillus thuringiensis. This is likely because spores are dormant bodies of bacteria, which are among the most stress-resistant and adaptable organisms that can survive adverse external environments such as extreme temperatures, absence of water or light, and presence of chemicals (Bağcıoğlu et al., 2019). Therefore, Bacillus can exist in large quantities when the soil’s micro-ecological environment is destroyed by continuous cropping. Bacillus also has many ecological functions such as enzyme production, salt tolerance, acid production, and phosphorus solubilisation (Céline et al., 2007). For example, Paenibacillus polymyxa secretes polypeptide proteins, enzymes, and plant hormones, which may be used to control plant diseases (Lin et al., 2018) and can promote plant growth by fixing nitrogen and dissolving phosphorus (Khan et al., 2008). Others, such as Bacillus amyloliquefaciens, secrete proteins, lipopeptides, and secondary metabolites to inhibit the growth of plant pathogenic bacteria (Yan et al., 2018). Streptomyces is the most diverse genus in the phylum of actinomycetes (Dai et al., 2014). It can adapt to different environments through a variety of self-produced secondary metabolites (Zeng et al., 2019; Ma et al., 2019). Therefore, of the 17 actinomycete species isolated in this study, 15 belonged to the genus Streptomyces. Additionally, Streptomyces can produce a variety of secondary metabolites such as aminoglycosides, nucleosides, polyenes, macrolides, hormones, and tetracyclines, which are important for cell wall and protein synthesis, plants growth, and the creation of an acidic environment that inhibits pathogens (Barakate et al., 2002; Wang et al., 2005; Hamedi and Mohammdipanah, 2015). Therefore, Streptomyces may be valuable for biological control of plant disease and promotion of plant growth. Among the six isolated fungi, Phytophthora, Fusarium solani, and Fusarium were the pathogenic microorganisms for Phytophthora capsica infection, root rot, and fusarium wilt, respectively, indicating that continuous cropping led to the domination of pathogenic fungi over beneficial bacteria. As a saprophytic fungus, aflatoxin produced by Aspergillus is widely distributed in the soil, which does not have a high requirement for growth and is an opportunistic plant pathogen (Horn, 2003). Penicillium oxalate promotes disease resistance by inducing plant resistance and phosphorus solubilisation (Fan et al., 2002; Peng et al., 2004; Ali et al., 2006). Penicillium expansum, on the other hand, can cause postharvest rot of apples and pears and even human intestinal diseases (Arici et al., 2000; Liu et al., 2010). We found that after continuous cropping, the majority of the bacteria other than Penicillium were pathogenic. The dominant bacterial strains isolated in this study, such as Bacillus, Streptomyces, and Penicillium, are beneficial and available microbial resources.

This research was executed using traditional isolation and culture methods. Compared with modern high-throughput molecular sequencing technologies, traditional methods lack versatility and comprehensiveness and can only be applied on cultivatable microorganisms in the soil, thus limiting the scope of the research (Yuan et al., 2014). However, modern molecular biology technology does not aim to obtain living microbial cells that can be cultured, making it difficult to accurately design and efficiently utilise these microorganisms to fine-tune microbial processes in the soil. In this aspect, the traditional pure culture technology can isolate and obtain pure microorganisms that may be directly or indirectly used for research, medicine, industry, and agricultural production through expansion and cultivation or transformation into new strains (Guo et al., 2006). In conclusion, our study provides a basis for human intervention to beneficially alter the soil microbial flora after continuous cropping to break the current limitations and sustainably guide agricultural production.

Supplementary Material

Supplementary materials are available at Horticultural Science and Technology website (https://www.hst-j.org).

Acknowledgements

This study was carried out with support from the Research Program for National Key and Development of China (grant no. 2017YFD 0201100), the Agricultural Research and Industrialization Project of Liaoning Province (grant nos. 2018103003 and 2018301003), Discipline Construction Project (2020DD082401) and Key Research and Development Project (2021HQ1903) of Liaoning Academy of Agricultural Sciences

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