Isolation of Trichoderma virens 6PS-2 and its effects on Fusarium proliferatum f. sp. Malus domestica MR5 related to apple replant disease in China

Apple replant disease(ARD) is primarily caused by biotic factors that seriously inhibits the development of apple industry. Therefore, the use of biological control measures to inhibit the main pathogens(such as Fusarium spp.) that cause ARD is of great significance to the sustainable development of the apple industry. Trichoderma virens 6PS-2, which exhibited antagonism toward a variety of pathogens, was screened from the rhizosphere soils of healthy apple trees(Malus robusta) in different replanted orchards in the Yantai and Zibo Cities, Shandong Province, China. Its fermentation extract inhibited the growth of pathogenic Fusarium proliferatum f. sp. Malus domestica MR5, which was proportional to the concentration. These substances also increased the hairy root volume and growth of Arabidopsis thaliana lateral roots. The phenotype of Malus hupehensis seedlings and microbial community structure in rhizosphere soils in greenhouse experiment using Highthroughput sequencing were analyzed, and the field experiment with grafted apple trees were used for further verification. Compared with the application of potato dextrose broth(PDB) medium, application of 6PS-2 spore suspension directly to replanted soils could improve the growth of M. hupehensis seedlings as well as the elongation of grafted apple trees. Concomitant decreases in the gene copy number of Fusarium and increases in the culturable bacteria/fungi were also observed in the greenhouse and field experiments. The abundance of Trichoderma,Bacillus, and Streptomyces increased significantly, but that of Fusarium, Pseudarthrobacter, and Humicola decreased. The content of esters, phenols,furans, and amino acids in root exudates of M. hupehensis seedlings increased, which significantly inhibited the multiplication of Fusarium, but was positively correlated with Bacillus and Trichoderma. In summary, T. virens 6PS-2 not only directly inhibits the activity of pathogenic Fusarium but also secrets secondary metabolites with antifungal and growth-promoting potential. In addition, 6PS-2 spore suspension can also promote the growth of plants to a certain extent, and change the soil microbial community structure of rhizosphere soils. It is believed that T. virens 6PS-2 has the potential for the alleviation of apple replant disease(ARD) in China.

Biochar alleviates apple replant disease by reducing the growth of Fusarium oxysporum and regulating microbial communities

Apple replant disease(ARD)negatively affects plant growth and reduces yields in replanted orchards.In this study,biochar was applied to apple replant soil with Fusarium oxysporum.Our aim was to investigate whether biochar could promote plant growth and alleviate apple replant disease by reducing the growth of harmful soil microorganisms,changing soil microbial community structure and improving the soil environment.This experiment included five treatments:apple replant soil(CK),methyl bromide fumigation apple replant soil(FM),replant soil with biochar addition(2%),replant soil with F.oxysporum spore solution(8×10^(7)spores·mL^(-1)),and replant soil with biochar and F.oxysporum spore solution addition.Seedling biomass,the activity of antioxidant enzymes in the leaves and roots,and soil environmental variables were measured.Microbial community composition and community structure were analyzed using 16SrDNA and ITS2 gene sequencing.Biochar significantly reduced the abundance of F.oxysporum and increased soil microbial diversity and richness.Biochar also increased the soil enzyme activities(urease,invertase,neutral phosphatase,and catalase),the biomass(plant height,fresh weight,dry weight)and the activity of antioxidant enzymes(superoxide dismutase,peroxidase,and catalase).The root indexes of apple seedlings was also increased in replant soil by biochar.In sum,biochar promoted the growth of plants,improved the replant soil environment,and alleviated apple replant disease.

Characterization of the microbial community response to replant diseases in peach orchards

This study attempted to monitor the development of microbial communities and reveal the correlation between the soil microbial community and soil nutrient factors over different years following the replanting of peach trees.The replanted soil(RS)and nonreplanted soil(NRS)were collected from peach orchards with different growth years(1,3,5,7,9,11,and 13 years)in the same region.The soil bacterial and fungal community diversities were analyzed by high-throughput sequencing technology.Redundancy analysis(RDA)was used to show the correlation between the soil microbial community and environmental variables.The alpha diversities of the bacterial and fungal communities indicated that RS contained a higher abundance of bacterial and fungal operational taxonomic units(OTUs)than NRS.NMDS and ANOSIM analyses showed that the soil bacterial and fungal communities were significantly(P<0.01)affected by planting years,and that the main changes occurred in the first and ninth planting years.The presence of the bacterial orders Sphingobacteriales,Burkholderiales and Actinomycetales changed significantly after replanting.Some bacteria associated with bioremediation,such as Burkholderiales and Intrasporangiaceae,and some harmful pathogens,such as Penicillium and Ophiostomatales,significantly increased after replanting(LDA score>3.0).In addition,the soil nutrient contents were lower in RS than in NRS in the early stage(1–5 years),and the RDA showed that bacterial and fungal phyla are closely associated with environmental variables,including the potential of hydrogen(pH),ammonium nitrogen(AN),available phosphorus(AP)and available potassium(AK).These results lead to a deeper understanding of the microbial responses to replanting in peach orchards.

Isolation of phloridzin-degrading,IAA-producing bacterium Ochrobactrum haematophilum and its effects on the apple replant soil environment

We isolated and identified a bacterium that could produce IAA and degrade phloridzin in the rhizosphere soil of healthy replanted apple(the rootstock is M9T337 and the scion is Yanfu 3),providing a theoretical basis for reducing the obstacles associated with apple replant disease(ARD).Isolates were screened using Salkowski colorimetry and screening medium for phloridzin.The isolate of interest(W6)was identified as Ochrobactrum haematophilum based on morphological analysis,physiological and biochemical tests,and 16S rDNA sequencing.In a laboratory experiment,W6 produced auxin and promoted the growth of Arabidopsis thaliana roots,and its degradation rate of 100 mg.L^(-1 )phloridzin was 62.0%.In a pot experiment,W6 significantly reduced the phenolic acid contents of replanted soil,lowered the abundance of the harmful fungus Fusarium solani,and increased soil enzyme activities,thereby improving the micro-ecological environment of replant soil.W6 increased the root antioxidant enzyme activity and leaf photosynthetic pigment content of replanted Malus hupehensis Rehd.seedlings,effectively alleviating the decrease in net photosynthetic rate,transpiration rate and stomatal conductance caused by ARD.In a field experiment,W6 also promoted the growth of replanted apple(the rootstock is M9T337 and the scion is Yanfu 3)saplings.Therefore,W6 can promote apple growth and degrade phenolic acids,and it can be used as an effective treatment for the reduction of ARD.

Silencing of early auxin responsive genes MdGH3-2/12 reduces the resistance to Fusarium solani in apple

Apple replant disease(ARD)has led to severe yield and quality reduction in the apple industry.Fusarium solani(F.solani)has been identified as one of the main microbial pathogens responsible for ARD.Auxin(indole-3-acetic acid,IAA),an endogenous hormone in plants,is involved in almost all plant growth and development processes and plays a role in plant immunity against pathogens.Gretchen Hagen3(GH3)is one of the early/primary auxin response genes.The aim of this study was to evaluate the function of MdGH3-2 and MdGH3-12 in the defense response of F.solani by treating MdGH3-2/12 RNAi plants with F.solani.The results show that under F.solani infection,RNAi of MdGH3-2/12 inhibited plant biomass accumulation and exacerbated root damage.After inoculation with F.solani,MdGH3-2/12 RNAi inhibited the biosynthesis of acid-amido synthetase.This led to the inhibition of free IAA combining with amino acids,resulting in excessive free IAA accumulation.This excessive free IAA altered plant tissue structure,accelerated fungal hyphal invasion,reduced the activity of antioxidant enzymes(SOD,POD and CAT),increased the reactive oxygen species(ROS)level,and reduced total chlorophyll content and photosynthetic ability,while regulating the expression of PR-related genes including PR1,PR4,PR5 and PR8.It also changed the contents of plant hormones and amino acids,and ultimately reduced the resistance to F.solani.In conclusion,these results demonstrate that MdGH3-2 and MdGH3-12 play an important role in apple tolerance to F.solani and ARD.

Effects of Soil Texture on the Growth of Young Apple Trees and Soil Microbial Community Structure Under Replanted Conditions

A two-year field experiment was carried out in order to study the occurrence degree and mechanism of apple replant disease(ARD)in the apple orchards with different soil textures.So we can adopt appropriate controlmeasures according to the severity of ARD.Healthy two-year-old seedlings with consistent growth were selected,of which the root stock was T337 and the scion was Yanfu 3.There were significant differences in biomass between methyl bromide fumigation and replanted treatments,and the difference was the largest in clay loam,followed by sandy loam,and loam,which verified ARD in clay loam was most serious,followed by sandy loam and loam.Based on high-throughput sequencing of fungi in soil samples,fungal richness and diversity were the highest in clay loam,followed by sandy loam,and loam.The relative abundance of Fusarium in SX,SL,FX,FL,WX and WL was 7.33%,19.32%,2.70%,4.24%,10.71%and 23.87%,respectively.Based on Real-time quantitative analysis,there were significant differences in the number of Fusarium oxysporum and Fusarium solani between methyl bromide fumigation and replanted treatments,i.e.,clay loam>sandy loam>loam.Fusarium was the main pathogen causing ARD.This shows that ARD is the most serious under replanted clay loam condition.High-throughput sequencing technology was used to prove the difference in Fusarium was one of the important reasons for ARD under different soil textures.This technology provides a new idea for the prevention and control of ARD.

The effects of crop rotation combinations on the soil quality of old apple orchard

This study investigated the effects of six crop rotation combinations on the soil quality of old apple orchard and seedling growth of Malus hupehensis Rehd.(apple rootstock) under pot conditions. The inhibitory effects of crops such as Allium fistulosum, Brassica juncea, and Triticum aestivum on four species of Fusarium were observed and compared in six treatments. These were continuous cropping(CK), fumigation with the methyl bromide(FM), rotating A. fistulosum only(R1), rotating A. fistulosum and T. aestivum(R2), rotating A. fistulosum, B. juncea, and T. aestivum(R3), and fallow(FC) in a year. The results showed that the biomass of Malus hupehensis Rehd. seedlings increased significantly. The root length increased and the root architecture was optimized. The respiration rate of the root system was increased by about 1 time after rotation. The treatments of R1, R2, R3, and FC increased bacterial count by 232.17%, 96.04%, 316.21%, and 60.02%, respectively. However, the fungi were reduced in varying degrees and bacteria/fungi ratio was increased by 5–10 times. The enzyme activities, p H, and organic matter were increased, but soil bulk density was decreased. Phenolic acids such as phloridzin was decreased significantly. The copy number of four Fusarium species declined by 85.59%, 74.94%, 69.68%, and 54.41% after rotating three different crops(R3 treatment). The root volatiles of three plants inhibited mycelial growth and spore germination of four Fusarium species.

Analysis of the Fungal Community in Apple Replanted Soil Around Bohai Gulf

Apple replant disease(ARD) is a frequently occurring plant disease in replanted orchards around Bohai Gulf, which causes growth inhibition and even death of plants. The aim of this study was to investigate the etiology of ARD around Bohai Gulf. In this study, the primary growth inhibition of apple seedlings was evaluated in ten replanted soils, sampled around Bohai Gulf. A fungal clone library was used to identify changes in the structure and composition of the soil fungal community. The results revealed that the Simpson diversity indices of Laizhou and Pulandian orchards were higher than others, presenting severe ARD. Ascomycota dominated around Bohai Gulf at the phyla level. Fusarium and Saccharomyces were abundant in all replanted soils. In addition, correlations between the relative abundance of fungal genera in soils and the severity of ARD were analyzed. The results showed that Fusarium was correlated positively with the severity of ARD, but Mortierella was negatively correlated. Furthermore, the quantitative PCR of Fusarium oxysporum, which was regarded as a factor of ARD, was performed. Overall, this study demonstrated that ARD was strongly associated with an unbalanced microbial ecosystem with more pathogenic fungi, while Fusarium in the apple replanted soil was the key factor for ARD around Bohai Gulf.