Identification And Biocontrol Efficacy Of Antagonistic Bacteria Against Stem Rot Of Cistanche Deserticola

 

Abstract

Objective: Stem rot is a major disease constraining the cultivation and industrial development of Cistanche deserticola. To obtain effective biocontrol strains and reduce problems associated with chemical control, this study aimed to provide microbial resources for biological control of stem rot in C. deserticola.

Methods: Antagonistic bacteria were isolated from soils collected in C. deserticola cultivation areas. Candidate strains with strong inhibition against three major pathogens-Fusarium oxysporum, Fusarium proliferatum, and Fusarium acuminatum-were directionally screened using a dual-culture plate confrontation assay. The selected strain was identified by combining morphological observation with molecular methods (16S rDNA sequencing and phylogenetic analysis). Extracellular enzyme production was assessed. Field trials were conducted to evaluate biocontrol efficacy.

Results: One highly effective antagonistic strain, WLKYLW-9, was obtained. The in vitro inhibition rates against F. oxysporum, F. proliferatum, and F. acuminatum were 74.36%, 64.62%, and 53.85%, respectively. In the field, the control efficacy reached 68.97%–77.71%. Strain WLKYLW-9 was identified as Bacillus halotolerans. Enzyme assays indicated that WLKYLW-9 secreted cellulase, protease, and amylase, suggesting that these enzymes may contribute to pathogen suppression.

Conclusion: WLKYLW-9 significantly inhibited the main pathogens causing stem rot of C. deserticola and shows substantial potential for development and application in biological control.

Keywords: Cistanche deserticola; stem rot; antagonistic bacteria; antifungal activity; control efficacy

 

1 Introduction

Cistanche deserticola (Rou Cong Rong) is a parasitic medicinal plant growing on the roots of halophytic desert plants and is known for tonic and endocrine-regulating functions. In recent years, after being included in the list of medicinal and edible homologous resources, market demand has increased markedly. With the maturation of artificial cultivation techniques, the industry has expanded rapidly. In Minqin County alone, the planting area has reached about 20,000 mu, and stem rot has gradually become the major barrier to sustainable development.

Based on isolation and identification of diseased samples collected from cultivation bases around Qingtu Lake, Xiqu Town, Minqin County, Wuwei City, Gansu Province, our group previously determined that the main pathogens in this region include F. proliferatum, F. oxysporum, and F. acuminatum [2]. Two typical symptom types are observed in the field: (1) early epidermal blackening and decay, with white flocculent mycelia under high humidity that later turn pink; and (2) initial dark brown to black spots, followed by overall darkening of the fleshy stem; the exterior remains firm without obvious soft rot, while internal tissues brown and eventually blacken [2].

This study focused on soils from the Qingtu Lake cultivation region and combined laboratory screening and field trials to select antagonistic bacteria with significant suppressive effects on stem rot, providing microbial resources and theoretical support for developing biopesticides and promoting sustainable production.

 

2 Materials and Methods

2.1 Materials

Soil samples: The experimental site was located in a C. deserticola cultivation base in the Qingtu Lake region of Minqin County, Wuwei City, Gansu Province. Thirty soil samples were collected.

Pathogens: Three pathogens previously isolated and identified by our group-F. proliferatum, F. oxysporum, and F. acuminatum-were preserved in the Key Laboratory of Fruit Tree Stress Physiology and Fruit Quality Regulation, Wuwei City [2].

Media: LB agar, NB broth, PDA, CMC medium, MH medium (casein medium), and ST medium (starch medium) were prepared according to the listed formulations. Media were adjusted to pH 7.0–7.2 and sterilized at 121 °C for 20 min.

Figure 1. Plate antagonistic effect of strain WLKYLW-9 against three pathogens causing stem rot in Cistanche deserticola.

Note: A represents the inhibitory effect of strain WLKYLW-9 on Fusarium oxysporum colonies; B represents normally growing Fusarium oxysporum colonies; C represents the inhibitory effect of strain WLKYLW-9 on Fusarium effusum colonies; D represents normally growing Fusarium effusum colonies; E represents the inhibitory effect of strain WLKYLW-9 on Fusarium argentis colonies; F represents normally growing Fusarium argentis colonies.

 

 

2.2 Instruments

Optical microscope (Olympus-Cx31rtsf, Olympus, Japan); constant-temperature incubator (DNP-9162BS-III, Shanghai Xinmiao); shaking incubator (ZWY-200D, Shanghai Zhicheng).

2.3 Methods

2.3.1 Isolation and screening of antagonists

Antagonistic bacteria were isolated from soil using the dilution plating method [3]. Screening was performed using dual-culture confrontation on PDA: 5-mm mycelial plugs of each pathogen were placed at the center of the plate, while ~1 mm³ blocks from bacterial single colonies were inoculated 2 cm from the plug at four symmetric points. Three independent plates were used as biological replicates for each treatment. Plates were incubated at 28 °C, inhibition zones were recorded, and inhibitory strains were re-screened. The inhibition rate was calculated as:

Inhibition rate (%)=Dc−DtDc×100\text{Inhibition rate (\%)}=\frac{D_c - D_t}{D_c}\times 100Inhibition rate (%)=Dc​Dc​−Dt​​×100

where DcD_cDc​ is colony diameter in the control and DtD_tDt​ is colony diameter in the treatment [4].

 

Table 1. Inhibition rate of strain WLKYLW-9 against three pathogens causing stem rot of Cistanche deserticola

Pathogen	Inhibition rate (%)
Fusarium oxysporum	74.36 ± 1.32ᵃ
Fusarium proliferatum	64.62 ± 0.78ᵇ
Fusarium acuminatum	53.85 ± 1.54ᶜ

Note: Different lowercase letters within the column indicate significant differences (P < 0.05).

 

Table 2. HC values of hydrolysis zones produced by strain WLKYLW-9 on different media

Medium	HC value
CMC	3.89 ± 0.06ᵃ
MH	2.33 ± 0.40ᵇ
ST	1.52 ± 0.22ᶜ

Note: Different lowercase letters within the column indicate significant differences (P < 0.05).

 

2.3.2 Extracellular enzyme assays

CMC, ST, and MH media were used to detect cellulase, amylase, and extracellular protease activities, respectively. Bacterial suspensions were adjusted to OD600_{600}600​=0.6. Each plate was spotted with: 1 μL fresh live cells, 1 μL sterile water, and 1 μL heat-killed cells (121 °C, 20 min), with an additional blank medium area as negative control. After incubation at 28 °C for 5 days, hydrolysis zones (clear halos) were assessed. CMC and ST plates were stained with Congo red and iodine, respectively [5]. Halo and colony diameters were measured using photos and ImageJ; the HC value was calculated as halo diameter/colony diameter [6].

2.3.3 Morphological and molecular identification

The selected strain was grown on NA at 28 °C for 24–48 h to observe colony morphology (color, size, margin, elevation). Gram staining, spore staining, and microscopy were performed. Genomic DNA was extracted using a commercial kit (SK8255, Sangon Biotech). The 16S rDNA gene was amplified using universal primers 27F and 1492R. PCR (25 μL) consisted of 2×Taq PCR Mix 12.5 μL, primers (10 μmol/L) 1 μL each, DNA template 1 μL, and sterile water 9.5 μL. Cycling: 95 °C 5 min; 30 cycles of 94 °C 30 s, 55 °C 30 s, 72 °C 90 s; final extension 72 °C 10 min. Products were verified by 1% agarose gel electrophoresis and sequenced bidirectionally (Sangon Biotech). After trimming and assembly, BLAST was used to identify homologous sequences in NCBI GenBank. Phylogenetic analysis was conducted in MEGA11 after MUSCLE alignment using the Neighbor-Joining method with 1,000 bootstrap replicates.

2.3.4 Field efficacy trial

Root-zone drench treatments were applied at the C. deserticola inoculation sites. Each treatment had 6 Haloxylon plants as biological replicates; each plant hosted 5–8 C. deserticola individuals. Treatments (drench order) were:

T1 (pathogen control): 200 mL pathogen spore suspension → 200 mL sterile NB → backfill sand → 5 L sterile water.

T2 (preventive antagonist): 200 mL antagonist fermentation broth → 200 mL pathogen spore suspension → backfill sand → 5 L sterile water.

T3 (curative antagonist): 200 mL pathogen spore suspension → 200 mL antagonist fermentation broth → backfill sand → 5 L sterile water.

Disease incidence was assessed 15 days after inoculation. Incidence (%) = (number of diseased roots/total investigated roots) ×100%. Control efficacy (%) = (incidence in control − incidence in treatment)/incidence in control ×100%.

Antagonist fermentation broth: cultured in NB at 28 °C, 150 rpm for 3 days; viable count adjusted to 1×1081\times 10^81×108 CFU/mL. Uninoculated NB served as blank. Pathogen spore suspension: Fusarium spp. cultured on PDA; spores were washed off, centrifuged, filtered, and adjusted to 1×1061\times 10^61×106 spores/mL for drenching. Spore germination rate was ≥90%. Each inoculation site received 200 mL (i.e., 2×1082\times 10^82×108 spores).

2.4 Data analysis

Data were analyzed using SPSS 16.0.

Figure 2. Clear hydrolysis zones produced by extracellular enzymes of strain WLKYLW-9

Note: A, CMC medium; B, MH medium; C, ST medium.

Figure 3. Colony morphology and Gram-staining microscopic observation of strain WLKYLW-9

Note: A, colony morphology of WLKYLW-9 after 7 days of incubation; B, Gram-stained cells observed under a 40× microscope.

3 Results

3.1 Isolation, screening, and antagonistic activity

A total of 121 bacterial isolates were obtained from 30 rhizosphere soil samples. Dual-culture screening against the three Fusarium pathogens identified WLKYLW-9 as the most effective strain. WLKYLW-9 significantly inhibited all three pathogens, with the strongest effect against F. oxysporum (74.36% inhibition), followed by F. proliferatum (64.62%) and F. acuminatum (53.85%) (Table 1). Under pathogen challenge, colony wrinkle distribution of WLKYLW-9 shifted, with fewer wrinkles near F. oxysporum and an outward shift under the other two pathogens (Fig. 1).

3.2 Extracellular enzyme activities

WLKYLW-9 grew well on CMC, MH, and ST media and produced clear hydrolysis zones, confirming secretion of cellulase, protease, and amylase. Larger halos were observed on CMC and MH, with HC values of 3.89 and 2.33, respectively (Table 2), indicating strong cellulase and protease activities. The halo on ST was smaller (HC 1.52) but clearly visible (Fig. 2).

3.3 Identification of strain WLKYLW-9

3.3.1 Morphological characteristics

On LB agar, colonies were milky white with moist surfaces and irregular margins; wrinkles appeared with prolonged incubation. Cells were Gram-positive rods producing endospores (Fig. 3), suggesting Bacillus spp.

3.3.2 Molecular identification

The 16S rDNA amplicon was 1,488 bp. BLAST and phylogenetic analysis (Fig. 4) showed 99% similarity to Bacillus halotolerans (GenBank accession NR115063), with the closest phylogenetic relationship. Based on morphology and molecular evidence, WLKYLW-9 was identified as B. halotolerans. The sequence was deposited in GenBank with accession OQ345825.

3.4 Field control efficacy

Field results (Table 3) demonstrated that the WLKYLW-9 liquid formulation significantly controlled stem rot caused by the mixed inoculum of F. oxysporum, F. proliferatum, and F. acuminatum. The pathogen control (T1) showed the highest incidence (87.20%), significantly higher than the preventive (T2) and curative (T3) treatments (P<0.05). T2 had the lowest incidence (19.44%), significantly lower than T3 (27.06%, P<0.05). Corrected control efficacy relative to T1 was 77.71% for T2 and 68.97% for T3, indicating better performance when applied preventively.

Table 3. Field control efficacy of strain WLKYLW-9 against stem rot of Cistanche deserticola

Treatment	Disease incidence (%)	Control efficacy (%)
T1	87.20 ± 0.73ᵃ	-
T2	19.44 ± 1.36ᶜ	77.71
T3	27.06 ± 8.15ᵇ	68.97

Note: Data are mean ± standard deviation (SD). Different lowercase letters within the same column indicate significant differences (P < 0.05).

 

4 Discussion

Antagonistic bacteria are widely used microbial resources for biological control and represent key sources for biopesticide development. Studies on antagonistic bacteria targeting stem rot of C. deserticola remain limited. Cheng et al. [8] suggested that Fusarium sambucinum may also be an important pathogen. In this study, a highly effective strain, WLKYLW-9, was isolated from soils in the Qingtu Lake region and identified as B. halotolerans.

Multiple studies support the biocontrol potential of Bacillus spp. For example, B. subtilis Bs916 achieved 55.6% control against tomato bacterial wilt [9], and B. mojavensis isolated from pear orchards showed significant efficacy against pear canker/dieback [10]. Biocontrol research on potato dry rot is more mature, with several broad-spectrum or pathogen-specific Bacillus strains reported, including B. amyloliquefaciens and B. velezensis [11]. Because stem rot of C. deserticola affects underground edible organs similarly to potato dry rot, biological control is particularly important.

 

Click the picture to get more details of organic cistanche base

B. halotolerans has been studied for both biocontrol and plant growth promotion. Yan et al. [12] isolated a strain from saline-alkali soil showing high antagonism against Staphylococcus aureus and Verticillium dahliae, likely via secondary metabolites or degradative enzymes, consistent with our findings. Zhu et al. [13] reported that B. halotolerans could enhance host resistance by increasing antioxidant enzyme activities. Here, WLKYLW-9 secreted cellulase, protease, and amylase-especially strong cellulase and protease-suggesting an important role in antagonism, in agreement with previous reports and our earlier work on pear disease [10,14]. Field trials further confirmed significant suppression of stem rot caused by mixed Fusarium pathogens. Since WLKYLW-9 was isolated from saline-alkali soils, its potential plant growth-promoting effects beyond disease control warrant further investigation.

 

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