Pathogen Identification and Indoor Fungicides Screening for Root Rot Disease in Tiller onion

doi:10.21203/rs.3.rs-2950844/v1

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Pathogen Identification and Indoor Fungicides Screening for Root Rot Disease in Tiller onion

https://doi.org/10.21203/rs.3.rs-2950844/v1

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This study aimed to identify the pathogen responsible for root rot disease in tiller onion in Nongan county, Jilin Province, China, and to screen for effective control measures using fungicides. The pathogen was isolated from diseased plants using the tissue block separation method and identified through morphological and molecular biological methods as Fusarium oxysporum. Indoor toxicity testing was conducted on 19 fungicides using the growth rate method, and the most effective agents were screened out. Results showed that nine agents had the best inhibitory effect on the pathogen, with EC₅₀ values of less than 0.1 mg/L. These agents include 99% propiconazole, 98% difenoconazole, 99% hymexazol, 98% myclobutanil, 98% prochloraz, 98% tebuconazole, 97% thiram, 70% thiophanate-methyl and 97% pyraclostrobine. Conversely, 99% sodium trisulfate and 98% copper calcium sulphate had the least inhibitory effect, with EC₅₀ values of more than 50 mg/L. The results provided a scientific basis for identifying and controlling root rot of tiller onion in Nongan County, Jilin Province, China.

Tiller onion

root rot

Fusarium oxysporum

fungicide screening

Tiller onion (Allium cepa var. aggregatum) is an annual herb belonging to the lily family and is a commonly cultivated onion and garlic vegetable in northern China. Tiller onion is a bush variety of onion that is also known as hairy onion, ghost onion or pearl onion (Cui C S and Fu X S 2000). The special flavor and ssential nutrients of tiller onions surpass those of regular onions. The bulbs contain volatile sulfides with a unique pungent flavor, flavonoids, phenolic compounds and a variety of nitrogenous compounds, which make tiller onions highly valuable for utilization in both eat freshly and processed characteristics (Liu S Y et al. 2018; Zhao J et al. 2015). Tillered onion has gradually become one of the emerging specialty vegetables in northeast China due to its distinct characteristics. It is a regional specialty vegetable that is widely grown in Heilongjiang and Jilin provinces, China, and is known as one of the “three spices” along with pepper and garlic. Tillered onions are widely exported to Southeast Asia, Russia, Japan and Korea (Zhao B H et al. 2019). As a result, bring substantial revenue to growers every year and playing a positive role in promoting increased production and income for farmers.

During the cultivation of tiller onions over many years, a root disease has been found that severely impacts their growth and development. This disease results in red discoloration of the root system, wilting of the plant, and rotting of the bulb disc, leading to a yield reduction of 30–40% in severe cases. Over time, the incidence of this disease has shown an increasing trend, seriously affecting the yield and quality of tiller onions and resulting in significant economic losses for farmers. Furthermore, this disease is hindering the development of the tiller onion industry. In order to identify the disease and to find out the cause of its occurrence. Despite an extensive review of the literature and information, no relevant reports pertaining to the type or cause of this disease were identified. Nevertheless, by analyzing similar diseases affecting onions of the same genus, onion root rot and stem base disc rot were found to be similar to this disease (Wei Z X et al. 2016; Zhao H X et al. 2019). Both diseases are caused by Fusarium spp. fungi and are typical soil-borne diseases that have devastated onion production and severely restricted the development of the onion industry (Zhao H X et al. 2019). The root disease affecting tiller onions was identified by utilizing the identification methods for onion root rot and stem-based disc rot. In 2022, plants infected with this disease were collected from the primary production area of tiller onion, Halahai Town, Nongan County, China. Screened for effective control agents through laboratory microscopy, isolation, identification and indoor pesticide screening. This research aims to provide a scientific and theoretical basis for the occurrence, as well as control methods.

Samples of tiller onion root rot disease were collected in June 2022 from Huaide Town, Gongzhuling County, and Halahai Town, Nongan County, Jilin Province, China, and brought back to the laboratory for isolation, purification and preservation. The fresh diseased samples with obvious symptoms were cut into small pieces of 0.5 cm x 0.5 cm, surface-sterilized by dipping in 75% ethanol for 60 s, 3% NaOCl for 3 min, and rinsing three times with sterile distilled water. Pieces were placed on potato dextrose agar (PDA) plates and incubated at 25 ± 1℃ for 4 days. After the mycelium had grown, it was purified 3 times by picking the edge of the colony to obtain a purified strain. The purified strains were transferred to PDA slant medium and stored in a 4°C incubator after the tubes were full grown.

For confirming pathogenicity, a spore suspension of 1×10⁶ spores/ml was prepared in sterile water and inoculated onto healthy Golden Dragon Pearl onion plants using the root irrigation method. For each treatment, five plants were injected with 10 ml of spore suspension. Control plants (n = 5) were injected with sterilized water. All plants were enclosed in plastic bags for 48 h in a greenhouse at 28℃ and 12 h/d light cycle. The morbidity survey was started after 7 days. The Koch's postulates was verified by reisolation from diseased plants to test if the pathogen could be isolated consistent with the inoculated pathogen.

Morphological and Molecular biology identification

Morphological characteristics of pathogens were examined with a microscope after incubation on PDA at 25°C with 12 h light/12 h dark period for 7 days. Subsequently, observe the colour and shape of the colonies, along with the shape, size and colour of the spores. For molecular support of these observations, the fungal genomic DNA was extracted from the surface mycelium of the pathogens using the Fungal Genomic DNA Rapid Extraction Kit (B516229-0050) and stored at -20°C. Segments of the internal transcribed spacer region of the rRNA gene ( ITS, primers ITS4 and ITS5), and translation elongation factor 1-alpha gene (TEF-1α, primers EF1 and EF2) were amplified by PCR (White, T. J. et al 1990; O’Donnell, K. and Cigelnik, E. 1997; O’Donnell, K., et al. 1997). The total PCR reaction system was 25 µL, including 1*Taq Mix 12.5 µL, 1 µL of each upstream and downstream primer, 2 µL of DNA template and 8.5 µL of ddH₂O. The PCR amplification conditions in the thermocycler were set as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, 35 cycles and termination at 72°C for 10 min. The amplification products were tested by agarose gel electrophoresis and then sent to biology company for sequencing. The DNA sequences from each of the isolates were used to search for sequence similarity against the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov).

Effect of the fungicides on mycelial growth of F. oxysporum

Determination of the inhibitory effect of different fungicides on pathogen using the growth rate method. The Nineteen fungicides used in this study are presented in Table 1. All fungicides were prepared as master mixes at a mass concentration of 10⁵ mg/L using the most suitable solvent. The fungicide master was diluted into six different concentration gradients using 0.1% Tween-80 and set aside. These concentration gradients were mixed with PDA medium to prepare plates at different dilutions (Table 1). The plates were uniformly punched and placed in the centre of the plate at the edge of the pathogenic colony. A blank control was created with a non-drug containing plate mixed with an equal volume of 0.1% Tween-80 solution, each treatment was repeated three times. The diameter of the colonies were measured using the crossover method when the control colonies reached 70 mm. The data were processed using the SPSS program and the regression equation of virulence, y = a + bx, was established via the least squares method. The effective medium concentration (EC₅₀) of each fungicides against the pathogen and the correlation coefficient, r values, were calculated. The effect of different fungicides on the mycelial growth rate of the test pathogens, their virulence, and the inhibitory effect of the fungicides were analysed and compared.

Table 1

List of Fungicides
Fungicides	Concentration (%)	Producers	Organic solvents	Concentration of plate (10³ mg/L)
propiconazole	99	Hebei Arltai Biotechnology Co., Ltd	Acetone	0.05, 0.25, 0.5, 1, 2.5, 10
picoxystrobin	99	Hebei Arltai Biotechnology Co., Ltd	Acetone	0.05, 0.25, 0.5, 1, 2.5, 10
fludioxonil	99	Hebei Arltai Biotechnology Co., Ltd	Acetone	0.5, 2.5, 5, 10, 25,100
prochloraz	98	Hebei Arltai Biotechnology Co., Ltd	Acetone	0.1, 0.5, 1, 2.5, 5, 20
phenazino-1-carboxylic acid	98	Hebei Arltai Biotechnology Co., Ltd	Acetone	0.1, 0.5, 1, 2.5, 5, 20
benziothiazolinone	99	Jiangsu Dekai Biotechnology Co., Ltd	Acetonitrile	0.05, 0.25, 0.5, 1, 2.5, 10
ifenoconazole	99	Shanghai Meitushi Biotechnology Co., Ltd	Acetonitrile	0.025, 0.05, 0.25, 0.5, 1, 5
validamycin	99	Hebei Arltai Biotechnology Co., Ltd	H₂o	0.05, 0.25, 0.5, 1, 2.5, 10
thifluzamide	99	Hebei Arltai Biotechnology Co., Ltd	Acetonitrile	0.1, 0.5, 1, 2.5, 5, 20
azoxystrobin	98.5	Shanghai Kunmiao Industrial Co., Ltd	Acetone	0.25, 1, 2.5, 5, 10, 50
pyraclostrobine	97	Shanghai Meitushi Biotechnology Co., Ltd	Acetone	0.05, 0.25, 0.5, 1, 2.5, 10
hymexazol	99	Hebei Arltai Biotechnology Co., Ltd	Acetone	0.5, 1, 2, 5, 10, 50
kasugamycin	99	Hebei Arltai Biotechnology Co., Ltd	Acetone	0.1, 0.5, 1, 2.5, 5, 20
myclobutanil	98	Shanghai Kunmiao Industrial Co., Ltd	Acetone	0.05, 0.25, 0.5, 1, 2.5, 10
tebuconazole	98	Shanghai Kunmiao Industrial Co., Ltd	Acetone	0.05, 0.25, 0.5, 1, 2.5, 10
fenaminosulf	99	Shanghai Meitushi Biotechnology Co., Ltd	Acetone	0.5, 2.5, 5, 10, 25, 50
thiram	97	Changshu Hengyao New Material Co., Ltd	Acetone	0.5, 1, 2, 5, 10, 50
thiophanate-methyl	70	Shanghai Meitushi Biotechnology Co., Ltd	Acetone	0.05, 0.25, 0.5, 1, 2.5, 10
copper calcium sulphate	98	Wujiang Yongsheng Chemical Co., LTD	H₂o	0.1, 0.5, 1, 2.5, 5, 20

Symptom of root rot of tiller onions and pathogen identification

Plants affected by this disease exhibit stunted growth, wilting, and drying of their above-ground parts (Fig. 1a). In addition, brown to red rot occurs on underground roots, and brown rot is observed on bulb discs (Fig. 1c). Affected plants show an increased number of tillers, but no expansion of onion heads (Fig. 1b), which severely affects the production of tiller onions. This disease can persist into the storage period and presents itself as dry rot of the shallot.

Confirming pathogenicity shown that after 10 days the inoculated plants showed similar symptoms to the original plants, with above-ground wilting and drying (Fig. 1e), underground roots not extending and appearing red and rotten (Fig. 1g), whereas the control plants had no symptom (Fig. 1d, f). Koch-type postulate was verified by successfully re-isolating the pathogen from symptomatic plants that exhibited same cultural traits as the inoculated pathogen, but not from the control leaves. It was proven that the pathogen isolated was indeed the causal agent of root rot in tiller onions.

Morphological characteristics of the isolate on PDA plates shown that the colonies initially appear white and grew in a scattered pattern. The mycelium surrounding the colonies was downy, and the hyphae were sparse (Fig. 1h). On the back of the plates, the colonies were pink or pale purple (Fig. 1i). Microconidia were observed to be hyaline, reniform or elliptic, and unicellular or bicellular, measuring 5.06 to 14.49 µm in length and 2.26 to 5.26 µm in width (Fig. 1j). The macroconidia were hyaline, falcate or fusiform, separated, and measured 15.8 to 49.5 × 2.55 to 6.06 µm (Fig. 1k). Based on these morphological and cultural characteristics, the causal agent was tentatively identified as Fusarium oxysporum.

Molecular biology techniques were employed for the identification of the pathogen, where both the ITS and TEF1-α regions were amplified and their sequences were deposited in GeneBank (accession ON725146 for ITS and ON725147 for TEF1-α). The ITS and TEF1-α sequences of strain T1 were compared to those in the GeneBank database, revealing a genetic similarity of 100% and 99% to F. oxysporum, respectively. A phylogenetic tree (Fig. 2) was constructed using MEGA 11 software, which demonstrated that strain T1 was most closely related to F. oxysporum and clustered in the same branch, thereby further confirming the taxonomic status of the causal agent. By combining both the morphological and molecular biological results, the pathogen caused tiller onion root rot was identified as F. oxysporum.

Effect of the fungicides on F. oxysporum

The impact of 19 different fungicides on the mycelial growth of F. oxysporum was assessed and the results are presented in Table 2. The results shown that the selected fungicides exhibited varying degrees of inhibitory effects on F. oxysporum, with significant differences in efficacy observed among the different fungicides. The antibacterial effect of propiconazole, difenoconazole, hymexazol, myclobutanil, prochloraz, tebuconazole, thiram, thiophanate-methyl and pyraclostrobine on the pathogen was significantly better than other experimental groups, with EC₅₀ less than 0.1mg/L. In contrast, sodium trisulfate and copper calcium sulphate had the worst bacteriostatic effect, with EC₅₀ values were greater than 50 mg/L. Secondly, the EC₅₀ and EC₉₀ values of the same fungicides were found to vary considerably in the study such as: picoxystrobin, thifluzamide, benziothiazolinone and phenazino-1-carboxylic acid. However, the difference in the EC₅₀ and EC₉₀ values of prochloraz and thiram was found to be insignificant, and the efficacy remained stable with EC₅₀ values less than 0.1 mg/L and EC₉₀ values less than 1 mg/L.

Table 2

Effect of 19 fungicides to mycelia of *F. oxysporum*
Fungicides	Toxicity equation	Correlation coefficient(R)	EC₅₀ (mg/L)	EC₉₀ (mg/L)
propiconazole	y = 0.2218x + 5.9153	0.9921	0.0001	44.8188
difenoconazole	y = 0.3156x + 5.9164	0.993	0.0012	14.3566
hymexazol	y = 0.2952x + 5.7869	0.9809	0.0022	47.3651
myclobutanil	y = 0.3692x + 5.9556	0.9255	0.0026	7.635
prochloraz	y = 1.3033x + 7.8083	0.9773	0.007	0.0674
tebuconazole	y = 0.7009x + 5.9569	0.9792	0.0431	2.9056
thiram	y = 1.0194x + 6.3509	0.8766	0.0473	0.8551
thiophanate-methyl	y = 0.6429x + 5.7348	0.9876	0.0719	7.0868
pyraclostrobine	y = 0.4324x + 5.4525	0.9898	0.0898	82.7067
picoxystrobin	y = 0.3245x + 5.2094	0.9495	0.2263	2011.9511
azoxystrobin	y = 0.6099x + 5.3235	0.8991	0.2948	37.2267
thifluzamide	y = 0.4824x + 5.1259	0.9777	0.5482	248.5844
benziothiazolinone	y = 0.4156x + 5.0532	0.9945	0.7446	903.043
phenazino-1-carboxylic acid	y = 0.3285x + 4.8871	0.954	2.2069	17597.2078
kasugamycin	y = 0.6098x + 4.4182	0.9337	8.9979	1137.0675
fludioxonil	y = 0.3388x + 4.5731	0.9032	18.1955	110254.876
validamycin A	y = 0.375x + 4.469	0.9897	26.0645	68147.5536
fenaminosulf	y = 0.5053x + 4.0728	0.9585	68.3719	23497.2421
copper calcium sulphate	y = 0.2316x + 3.5977	0.9397	1131370.812	3.85404E + 11

The pathogen causing root rot of tiller onions has been identified as F. oxysporum. F. oxysporum is a broad-spectrum pathogen known to cause root diseases in a wide range of plants. However, F. oxysporum causing root rot of tiller onion is the first report in both Chinese and international literature. Previous studies have reported only F. oxysporum as causing disease during both the storage and growth stages of onion cultivation (Wei Z X, et al. 2016; Su J H, et al. 2016; Xu D, et al. 2010). Further research will focus on determining whether this pathogen can survive in storage and cause disease during that time, and acting as a primary source of infection in the subsequent year.

The present study screened different fungicides for their ability to inhibit the pathogen through indoor agent screening. Among the fungicides tested, propiconazole, difenoconazole, hymexazol, myclobutanil, prochloraz, tebuconazole, thiram, thiophanate-methyl and pyraclostrobine exhibited promising results, which are consistent with the findings of other researchers. previous studies have demonstrated that propiconazole is highly effective against F. oxysporum on onion and potato (Su J H, et al. 2016; Ning N N, et al.2021). Whereas myclobutanil and prochloraz were found to be more effective than other fungicides in inhibiting F. oxysporum on melon through indoor virulence tests and pot experiments (Gu M L, et al. 2019). Pyraclostrobine was also found to have better inhibition of F. oxysporum on prunus and alfalfa by Xiao Rongfeng and Jing Xuemin (Xiao R F, et al. 2020; Jing X M, et al. 2019. Secondly, the EC₅₀ and EC₉₀ values of certain fungicides, including picoxystrobin, thifluzamide, benziothiazolinone and phenazino-1-carboxylic acid, varied considerably in this study. However, the difference between the EC₅₀ and EC₉₀ values of prochloraz and thiram is not significant and the efficacy is stable. It is recommended to use stable fungicides or alternate between fungicides when selecting control agents.

Tiller onion is an important regional specialty vegetable in northern China, however, there is currently a lack of research on root diseases of tiller onion. The discovery of root rot in tiller onion has significant implications for the control of tiller onion diseases. The pathogen produces a large amount of toxins in the process of infesting the roots, which severely hindering the normal growth and development of tiller onion, leading to a reduction in its quality and yield. As a result, it directly causes significant economic losses for producers. This study provides a theoretical basis for controlling root rot in tiller onion and provides selective fungicides that can be used for controlling root rot effectively.

Acknowledgement We are grateful to all the providers of tiller onion root rot disease.

Funding This study was supported by Jilin Provincial Science and Technology Development Plan (2021102063NC), Jilin Province, and China Agriculture Research System of MOF and MARA (CARA-24-G-05).

Compliance with ethical standards

Conflicts of interest We have no conflict of interest.

Human and animal rights statement This article does not contain any studies with human participants or animals.

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Pathogen Identification and Indoor Fungicides Screening for Root Rot Disease in Tiller onion

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Abstract

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Introduction

Materials and methods

Morphological and Molecular biology identification

Results

Symptom of root rot of tiller onions and pathogen identification

Discussion

Declarations

References

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