Characterization of Fungal Community Composition in the Rhizosphere Soil of Actinidia chinensis Planch Accompanied by Root Rot

Nowadays, with the development of Actinidia chinensis Planch cultivation, occurrence of Actinidia chinensis Planch root rot disease have increasingly affected the yield and quality of Actinidia chinensis Planch which caused by many kinds of fungal communities. The aim of this paper is to study fungal community structure in rhizosphere soil of Actinidia chinensis Planch infected and non- infected by root rot disease, and to look for monitoring of Actinidia chinensis Planch root rot disease and green ecological control. The rhizosphere soil samples of Actinidia chinensis Planch infected and non-infected by root rot disease were collected respectively in western mountain areas of Huangyan, Taizhou city, Zhejiang province,China, which is the one of the largest Actinidia chinensis Planch planting regional of Zhejiang province. The fungal community structure of rhizosphere soil of Actinidia chinensis Planch were determined by high throughput sequencing techniques. Compared with non-infected soil, the Chao1 indexes of fungal in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were decreased with significant level. At phylum level, the relative abundance of some dominant fungal in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were lower than those of non-infected with significant level(P<0.01), such as Basidiomycota, Fungi-unclassified and Zygomycota, while the relative abundance of Ascomycota in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were higher than those of non-infected with significant level(P<0.01). At genus level, the relative abundance of some dominant fungal in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were higher than those of non-infected with significant level, such as Gibberella, Nectria, Fusarium, Pseudogymnoascus and Hypocrea, while the relative abundance of some fungal in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were lower than those of non-infected with significant level(P<0.01), including Agaricomycetes-unclassified, Cladorrhinum, Trechispora, Ophiocordyceps, Acremonium, Ascobolus and Mortierella. It is conclude that in rhizosphere soils of Actinidia chinensis Planch infected with root rot disease, At the phylum and genus levels, great variations in the abundance of the dominant fungal have been confirmed. Actinidia chinensis Planch root rot disease is closely related to the structure of rhizosphere fungi community, and the changes of the balance and diversity of soil fungi is the major cause of Actinidia chinensis Planch root rot disease. The finding could provide some theoretical reference for controlling Actinidia chinensis Planch root rot disease in Zhejiang Province.


Introduction
The rhizosphere soil is the area of soil surrounding plants roots.It is areas for microbial interactions because plants roots provide exudates that are the main food source for microbe.The soil rhizosphere is the place where plants , soil, and microbe interact actively.The great amount of microbe are playing a key roles in maintaining balance of ecology system.Some microbe of the rhizosphere community have deleterious or benefical effects on plants.Some soil microorganisms participate in detrimental interactions with crops plants, resulting in root rot disease, while others have benefit effects on crops by promoting the growth and development of crop plants and controlling soil borne disease.So it is important to reveal the information about microbial community structure to understand the roles and significance of difference microbe of rhizosphere soil in biological controlling soil borne diseases.
Illumina Sequencing features greater capacity of estimation of microbial community structure and diversity.ZHAO et al has studied the differences of the composition and diversity of fungal community in the rhizosphere soil of healthy and infected potato plants of soil fungal community with the occurrence of verticillium wilt using Illumina sequencing technology.The results showed that the relative abundance of Ascomycota and Mortierellomycota in soils of infected plants was increased by 20.68% and 16.16%, respectively [1] at genus level (ZHAO et al .,2021).YANG G Z et al has studied the effects of apple root rot disease on rhizosphere soil microbe in an apple orchard based on Illumina Sequencing technology .The results showed that fungal shannon and Chao 1 index in disease soil were significantly higher than in healthy soil, while Simpson index was lower than that with healthy soil.Ascomycetes fungi in diseased soil were significantly lower than those in healthy soil at phylum level(YANG G Z et al ,2020).But there existed few reports about the effects of root rot diseases on the fungal communities in rhizosphere soil of Actinidia chinensis Planch plants.
Root rot diseases caused mainly by fungal pathogens have been common in Actinidia chinensis Planch planting fields( BI X Q et al, 2020), which is a native species of Yichang city,Hubei province, China.It is distributed mostly in many province such as Shaanxi, Hunan , Hubei, Jiangsu, Guangdong, Guangxi ,China.Actinidia chinensis Planch is known as health food because of its rich nutritional components of vitamin C, which is benifical to health of human being.Now China has become the worlds largest Actinidia chinensis Planch producer and plantable country.In these days, there have been decreases in yields and quality because of root rot of Actinidia chinensis Planch plants caused by fungal diseases.But few effective biological contral approach have been proposed to control root rot disease that affect the quality and yields of Actinidia chinensis Planch under fields.So it is necessary to reveal the rhizosphere soil fungal composition and diversity associated with Actinidia chinensis Planch plants infected with root rot under fields conditions for proposing effective biological control method.
In this paper, Illumina-based high throughput sequencin was employed to profile the main fungal rhizosphere taxa associated with Actinidia chinensis Planch plants infected with root rot under fields conditions , determing whether the fungal community composition differs for healthy and root rot plants to faciliate microbiol control for root rot disease of Actinidia chinensis Planch plants under fields conditions for proposing effective biological control methods( WU Z X et al ,2015).

Site Information
The study region is situated at Actinidia chinensis Planch plantation in Jiangshan city ,Zhejiang province, China.The study region features monsoonal subtropical climate, with average rainfall of 1648.11mm and average air temperature 17.11℃,respectively.The frost-free period there is 249.7days.The soil type there is sandy loam.The variety of Actinidia chinensis Planch is "Hongyang".The soil pH there is 6.5, soil organic matter is 19.1689g/kg.Soil available K is 158.436mg/kg.Soil available P is 69.051mg/kg.Soil total N is 1.095g/kg.Soil bulk density is 1.215 g/cm 3 .

Sample collection
Three plots with the area of 10m ×60 m at a 5m internal distance were selected in Actinidia chinensis Planch plantation .Each plot has the same soil type with consistant fertilization and other agricultural practices.In each plot, three Actinidia chinensis Planch trees infected with root rot diseases were randomly choosed in june 18 ,2021, which show the similar symptoms of root rot diseases .Three Actinidia chinensis Planch trees with healthy were also randomly choosed as control.Rhizosphere soils were obtained from healthy Actinidia chinensis Planch plants and from Actinidia chinensis Planch plants infected with root rot diseases(.Chen H S et al. 2022).

DNA extractions
DNA from different samples was abstracted employing the E.Z.N.A. ®Stool DNA Kit(D4015, Omega, Inc., USA) in the light of manufacturer's instructions.DNA extracted for 30 times from every treated soil sample was mixed as one DNA mixed sample.Then, the three mixed DNA samples were used for the fungal internal transcribed spacer (ITS) gene amplification.The total DNA was eluted in 50 μL of Elution buffer and stored at -80 °C until measurement in the PCR by LC-Bio Technology Co., Ltd, Hang Zhou, Zhejiang Province, China.Tip:The kit used by sample type .

Fungal ITS gene amplification and Illumina sequencing
We consulted the protocol described by CAPORASO J G et al( CAPORASO J G et al,2012) .To study the diversity and composition of the fungal communities in rhizosphere soil under healthy and infected is Planch.Primers ITS5-1737F and ITS2-2043R were selected to amplify the ITS1 region of the fungal genes.DNA was amplified following a previously described protoco(MAGOC T et al,2011).

Data analysis
Soil samples were sequenced on an Illumina NovaSeq platform in the light the manufacturer's instructions, supplied by LC-Bio.Paired-end reads was assigned to samples in the light of their unique barcode and truncated by shortening the barcode and primer sequence.Paired-end reads were unified using FLASH.Quality filtering on the raw reads were performed under specific filtering conditions to obtain the high-quality clean tags in the light of the fqtrim(v0.94).Chimeric sequences were screened employingVsearch software(v2.3.4).After dereplication employing DADA2,we acquired feature table and feature sequence.Alpha diversity and beta diversity were computed by QIIME2,which the same number of sequences were purified randomly through decreasing the number of sequences to the low limit of some samples, and the relative abundance is employed in taxonomy.Alpha diversity and Beta diversity were studied by QIIME2 process, and pictures were created by R (v3.5.2) .The sequence alignment of species annotation was conducted by QIIME2 plugin feature-classifier, and the alignment database was SILVA and NT-16S( LOGUE J B et al,2016)

Sequencing data analysis
The large number of effective sequences were acquired from the samples sequenced using Miseq at each classification leve(Table 1)l.The effective sequences obtained from two kinds of samples in this regions were 79197 and 84397 respectively.After removal of chimeras, 97% of the effective sequences were clustered as OTUs to obtain the abundance information of fungal in rhizosphere soils of root rot and healthy Actinidia chinensis Planch plants.The soil samples from rhizosphere soils of root rot Actinidia chinensis Planch plants had the number of OTUs with 479, while the soil sample from rhizosphere soils of healthy Actinidia chinensis Planch plants had the number of OTUs with 587(Fig.1).The OTUs in soil samples from root rot Actinidia chinensis Planch plants is significantly lower than that from healthy plants (18.40%).

Taxonomic composition of fungal community at the phylum level
The overall fungal compositions of the samples were similar, but some groups of fungal communities in the rhizosphere soil sample from the healthy and root rot of Actinidia chinensis Planch comprised different proportions at the phylum level(Fig.3).The results showed that Illumina high-throughput sequencing produced few reads those were unclassified, but most of the sequence reads were identified as fungal belonging to 9 phyla.The fungal representative phyla such as Ascomycota,Basidiomycota, Zygomycota.Ascomycota was the most abundant phylum in all samples, accounting for 44.76-96.55% of the total valid reads in all samples.Basidiomycota was the second most abundant phylum in all samples.Zygomycota was the third most abundant phylum in all samples.
Compared with the healthy samples from the same places, the root rot samples comprised relative lower percentages of Basidiomycota , unclassified fungal and Zygomycota (94.66,83.03and 83.93%, respectively),but relatively higher percentage of Ascomycota(97.31%).

Figure 3
The fungal community in rhizosphere soil of Actinidia chinensis Planch at phylum level.Taxonomic composition of fungal community at genus level The overall fungal compositions of soil samples were similar, but some groups of fungal communities in the rhizosphere soil sample from the healthy and root rot of Actinidia chinensis Planch contained different proportions at genus level(Fig.4).At genus level, the most frequent genus were Gibberella,Nectria,Cladorrhinum,Trechispora,Fusarium,Ophiocordyceps,Pseudogymnoascus,Acremo nium,Hypocrea,Ascobolus,Haematonectria.Compared with the healthy soil samples from the same sites, the samples from rhizosphere soil from root rot Actinidia chinensis Planch contained relative higher percentages of Gibberella, Nectria, Fusarium, Pseudogymnoascus, Hypocrea (P<0.01), but relative lower percentages of Cladorrhinum,Trechispora,Ophiocordyceps,Acremonium,Ascobolus (P<0.01).So it can be concluded that the original antagonistic equilibrium of microorganisms in rhizosphere soil of Actinidia chinensis Planch plants was destroy because of infection with root rot disease.

Discussion
A large number of greatly diverse soil microbe are associated with plants roots, which they play important parts in plant growth and health.The results of our experiment showed that the healthy rhizosphere soil samples contained relatively lower percentages of Ascomycota (115.71%)compared with the root rot soil samples, relative higher percentages of Basidiomycota and Zygomycota (94.66% and 83.93%) respectively at phylum level.XIE Y Q et al has conducted the experiments to show that Ascomycota and Zygomycota were the main phyla in rhizosphere soil of diseased garlic, and the high levels of Ascomycota were closely related to the occurrence of garlic root rot disease(XIE Y Q et al, 2020).Our results are consistent with their results, indicating that the root exudates in healthy Actinidia chinensis Planch plants can suppress the growth and development of Ascomycota , promote the growth and development of Zygomycota and Basidiomycota.
Soil Mortierella is probiotic fungal flora, which can produce many kinds of bioactive substances.It has strong ability to degrade many kinds of soil organic pollutants (ELLEGAARD J L et al ,2013;PAPANIKOLAOU S et al ,2019).Our results showed that the relative abundance of Mortierella in rhizosphere soil of root rot samples is lower than that of healthy samples.Moreover, the relative abundance of Cladorrhinum, Trechispora, Ophiocordyceps in rhizosphere soil of root rot samples is lower than those of healthy samples, which have ability to biological control diseases.The relative abundance of Fusarium in rhizosphere soil of root rot samples is higher than that of healthy samples, indicating that it is one of the main phthogens causing root rot of Actinidia chinensis Planch plants.

Conclusion
Compared with non-infected soil, the Chao1 indexes of fungal in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were decreased with significant level.At phylum level, the relative abundance of some dominant fungal in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were lower than those of non-infected with significant level(P<0.01),such as Basidiomycota, Fungi-unclassified and Zygomycota, while the relative abundance of Ascomycota in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were higher than those of non-infected with significant level(P<0.01).At genus level, the relative abundance of some dominant fungal in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were higher than those of non-infected with significant level, such as Gibberella, Nectria, Fusarium, Pseudogymnoascus and Hypocrea, while the relative abundance of some fungal in rhizosphere soil of Actinidia chinensis Planch infected by root rot disease were lower than those of non-infected with significant level(P<0.01),includingAgaricomycetes-unclassified, Cladorrhinum, Trechispora, Ophiocordyceps, Acremonium, Ascobolus and Mortierella.It is conclude that in rhizosphere soils of Actinidia chinensis Planch infected with root rot disease, At the phylum and genus levels, great variations in the abundance of the dominant fungal have been confirmed.The changes of the balance and diversity of soil fungi is the major cause of Actinidia chinensis Planch root rot disease.

Funding Information
Research on Improving the Employment Competitiveness of Vocational Electromechanical Students under the Background of Industry Education Integration, 2023 Zhejiang Province Zhonghua Vocational Education Research Project, (ZJCV2023C30)

Figure 4 .
Figure 4.The fungal community in rhizosphere soil of Actinidia chinensis Planch at genus level.

Table 1 .
The sequence readings OTUs of fungal in rhizosphere soils of Actinidia chinensis Planch under infected and non-infected with root rot disease

Table 2
Comparison of alpha diversity of fungal communities in rhizosphere soils of Actinidia chinensis Planch under infected and non-infected with root rot disease