Seedling Petri-dish inoculation method:A robust,easy-to-use and reliable assay for studying plant-Ralstonia solanacearum interactions

Ralstonia solanacearum causes a lethal bacterial wilt disease in many crops,leading to huge losses in crop production every year.Understanding of plant-R.solanacearum interactions will aid to develop efficient strategies to control the disease.As a soilborne pathogen,R.solanacearum naturally infects plants via roots.A huge limitation in studying plant-R.solanacearum interactions is the large variation of R.solanacearum infection assay due to the variable soil conditions and uneven inoculum exposure.Here,we developed a robust and reliable Petri-dish inoculation method which allows consistent and stable infection in young plant seedlings.This method is easy to use,takes about only 10 days from seed germination to the completion of inoculation assay,and requires less inoculum of bacteria as well as growth chamber space.We proved the efficacy of the seedling Petri-dish inoculation method by analyzing plant defense primed by molecular patterns,resistance of defense-related plant mutants,and virulence of R.solanacearum mutants.Furthermore,we demonstrated that the seedling Petri-dish inoculation method can be applied to other host plants such as tobacco and has great potential for high-throughput screening of resistant plant germplasms to bacterial wilt in the future.

Influence of different inoculation methods on the evaluation of the resistance to rice sheath blight(Rhizoctonia solani Kuhn)

To establish a standard system for geneticstudies on sheath blight resistance, a field testwas conducted at the experimental farm ofYangzhou University to compare several pro-cedures for inoculating rice plants with R.solani Kuhn (RH9). The varieties used wereJasmine 85, Teqing (resistant or moderatelyresistant), and Lemont (susceptible). They

Construction and Application of a High-throughput Method for Identifying Maize Resistance to Aspergillus flavus in the Field

In order to establish a fast and effective high-throughput method for identifying maize resistance to Aspergillus flavus infection in the field,5 conidial concentrations and 3 inoculation methods were compared and optimized. A correlation model between disease levels and aflatoxin B1 accumulation was established. The results showed that inoculating maize inbred lines with the optimal conidial concentration of 1.0×10^6 c/mL could accurately show the different resistance levels of maize to A.flavus infection,and the spiral quantitative inoculation technique could effectively avoid the defect of calculation error caused by commonly used methods. The positive correlation model of resistance level and aflatoxin B1 accumulation could effectively predict the capability of maize being resistant to A.flavus. 8 resistant and 9 medium resistant maize inbred lines of the RIL population were identified.The spiral quantitative identification method realized the high-throughput identification of maize inbred lines resistant to A.flavus infection in the field,and the results have shown high accuracy,stability and repeatability.

Effect of Different Inoculation Methods on Pathogenicity of Fusarium proliferatum to Alfalfa

[Objectives]To find out the suitable inoculation method for Fusarium proliferatum.[Methods]Longmu 801 and TG4 were inoculated with rice grain-based inoculum at the bud and seedling stages and with root irrigation at the seedling stage,respectively,and then,the plant height,aboveground biomass,underground biomass,incidence rate and disease index of the alfalfa were determined to conduct comprehensive resistance evaluation.[Results]There was obvious difference in disease resistance between the two alfalfa varieties.Among the three inoculation methods,the relative plant height,relative aboveground biomass and relative underground biomass of Longmu 801 differed insignificantly(P>0.05),all around 100.There were significant differences in TG4 among different inoculation methods(P<0.05).The incidence rates and disease indices of Longmu 801 and TG4 inoculated with rice grain-based inoculum at the bud stage were the highest,and their incidence rates and disease indices were 73.3%and 100%,and 40 and 60,respectively,significantly higher than those under the other two inoculation methods.The incidence rates and disease indices of Longmu 801 and TG4 inoculated with rice grain-based inoculum at the seedling stage were the lowest,and their incidence rates and disease indices were 0%and 13.30%,and 0 and 5.33,respectively,significantly lower than those under the other two inoculation methods.[Conclusions]The inoculation time and inoculation method of the pathogen both affect the inoculation effect.Among the three inoculation methods,the inoculation with rice grain-based inoculum at the bud stage is the most pathogenic to alfalfa seedlings,and can be popularized in the resistance evaluation of alfalfa to root diseases.

Establishment and Application of Identification Methods for Resistance to Sugarcane White Leaf Disease(SCWL)

[Objectives]This study was conducted to establish simple, efficient, stable, standardized and practical identification methods for sugarcane resistance to white leaf disease(SCWL), and promote the breeding for sugarcane resistance to SCWL. [Methods]The identification technology of sugarcane resistance to SCWL was systematically studied and explored from the aspects of sugarcane material treatment and planting, inoculation liquid preparation, inoculation method, disease investigation, grading standard formulation, etc., and two sets of simple, efficient, stable, standardized and practical accurate identification methods for sugarcane resistance to SCWL were created for the first time, namely, the seed cane coating inoculation method and the stem-cutting inoculation method at the growth stage. The seed cane coating inoculation method includes the steps of directly screening SCWL phytoplasma, extracting juice from cane and adding 10 times of sterile water to prepare an inoculation liquid, spraying seed cane on plastic film to keep moisture, planting the inoculated materials in barrels in an insect-proof greenhouse for cultivation, investigating the incidence rate 30 d after inoculation, and evaluating the disease resistance according to the 1-5 level standard. The method of stem-cutting inoculation includes the steps of directly screening sugarcane stems carrying SCWL phytoplasma and adding 10 times of sterile water to prepare an inoculation liquid, cultivating the identification materials in an insect-proof greenhouse, dropping 100 μl of the inoculation liquid into each root incision with a pipette gun at the age of 6 months, investigating the incidence rate 20 d after planting, and evaluating the disease resistance according to the 1-5 level standard. [Results] The two methods are similar to the natural transmission method. After inoculation, SCML occurred significantly, with high sensitivity and good reproducibility. The results of resistance identification were consistent with those of natural disease in the field. Through the two inoculation methods and field natural disease investigation, the resistance of 10 main cultivars to SCML was identified, which was true and reliable. [Conclusions] This study can provide standard varieties for identification of SCML resistance in the future.

Pathogenicity of Septoria carvi Syd. towards Caraway Carum carvi L, （Apiaceae）

Septoria carvi belong to the fungi recently detected on caraway plants in the Polish climatic conditions. In many European countries, this fungus causes significant losses in caraway cultivation and decreases the quality and quantity of the herb material. The aim of the present work was to study the fungus harmfulness towards the sowing materials, stems, leaves and umbels of caraway. The pathogenicity tests according to Koch＇s postulates were carried out for isolates ofS. carvi using post-culture liquids and a water suspension of conidia to inoculate the plants＇ parts. The studies were conducted in the laboratory, the climatic chamber and in field conditions. S. carvi caused disease symptoms in the form of spots and necrosis on all tested plants organs. The infection was followed by natural openings what was shown by scanning electron microscope （SEM） microscopic observations. The efficiency of inoculation increased injuries and anatomical dipping of plant epidermal tissue. The study showed the ability for asymptomatic S. carvi colonization of inoculated plants＇ parts. The performed pathogenicity tests and microscopic observations SEM showed that the tested isolates of S. carvi were occasional pathogens of caraway.

Root Plate Growth in Sunflower and Its Relevance to Sclerotinia Basal Stalk Rot

Sclerotinia basal stalk rot (BSR) of sunflower (Helianthus annuus L.) is a fungal disease of the roots that causes symptoms of wilt and a basal stem lesion. Evaluating root plate growth could improve our understanding of BSR. Separate studies were conducted to determine the effect of sunflower growth stage or genotype on root plate diameter in North American environments. Root plate diameter of cultivated hybrids at reproductive growth stages was 3 to 4 times larger than vegetative stages. Cultivated hybrids had larger root plate diameter than interspecific lines. These results have implications for artificial inoculation methods that evaluate genotypes for BSR resistance in the field or greenhouse. Disease escapes can occur if field-grown plants are inoculated too far from the root plate and/or too early at vegetative growth stages. Side-dressing mycelium-infested cereal grain closer (i.e., 10 cm) to plants at reproductive growth stages (i.e., R1 - R4) can increase disease pressure and reduce disease escapes. These guidelines for the field can be used to validate results from artificial inoculations in the greenhouse.

In-vitro vs in-vivo Inoculation: Screening for Resistance of Australian Rice Genotypes Against Blast Fungus

To assist with rapid screening for rice blast resistance as a precursor in a breeding program, the susceptibility to rice blast of 13 rice genotypes from Australia was evaluated in May to June 2013 using three distinct inoculation methods（spot, filter paper and standard methods） at seedling, vegetative and reproductive stages. The results revealed that the spot and filter paper inoculation methods were successful in discerning susceptibility to the rice blast disease（P ≤ 0.05）. Disease susceptibility declined significantly from the vegetative to reproductive stages. The standard method was conducted at three different stages for pot plants grown inside the mist house. However, low temperatures did not produce disease symptoms except in a few genotypes. Among the 13 rice genotypes screened, AAT9 expressed a highly resistant response, and AAT4, AAT6, AAT10, AAT11, AAT13, AAT17 and AAT18 expressed resistance at various stages. The results will be useful for selecting elite genotypes for disease tolerance where rice blast is prevalent. In addition, the resistant genotypes can serve as a gene pool used in breeding programmes to develop new resistant genotypes.