Maize ear rot severely impacts maize yield and quality, cultivating and planting resistant varieties is the most economical and effective prevention measure. Currently, the breeding of resistant varieties for maize ear rot mainly focuses on the severity of disease occurrence, while the accumulation of toxins within the kernels after disease occurrence is easily overlooked. This study aims to establish a resistance evaluation system to Fusarium ear rot based on fumonisin content, and to provide a basis for the breeding of new maize varieties with resistance to toxin production.

The F. verticillioides with GFP-labeled was inoculated into the ears of Zhengdan 958 using the acupuncture inoculation method, then the infection and toxin production of the pathogen on the ears were investigated. The F. verticillioides was inoculated into 36 main cultivated maize varieties, and their resistance was evaluated according to the current standard, the fumonisin content in kernels of each variety was determined by enzyme-linked immunosorbent assay (ELISA), the correlation between the fumonisin content and the average disease grade was analyzed, and a resistance evaluation system for maize varieties against Fusarium ear rot was constructed based on the fumonisin content ratio. Using this evaluation system and the current industry standard simultaneously, the resistance of 142 inbred lines to Fusarium ear rot was evaluated.

After the maize ears were infected by F. verticillioides, in addition to the visibly moldy kernels, there were also kernels that were symptomless but still infected. These asymptomatic kernels not only affect the judgment of the severity of ear rot occurrence, but also increase the content of fumonisin in kernels. The fumonisin content in kernels was moderately correlated with the average disease grade (r=0.39), indicating that it is impossible to select resistant varieties against fumonisin production based solely on the average disease grade. Therefore, the resistance evaluation system for maize varieties to Fusarium ear rot was established based on the fumonisin content ratio in kernels: the fumonisin content ratio<0.45, highly resistant (HR); 0.45≤the fumonisin content ratio<0.65, resistant (R); 0.65≤the fumonisin content ratio<0.85, moderately resistant (MR); 0.85≤the fumonisin content ratio<1.25, susceptible (S); the fumonisin content ratio≥1.25, highly susceptible (HS). Among the 142 inbred lines, the concordance rate between the two evaluation methods was only 14.79%, but 37.32% of the inbred lines showed moderate or higher resistance in both evaluation methods.

Based on the fumonisin content ratio in kernels, a new system for evaluating the resistance of maize varieties to Fusarium ear rot was established. The concordance rate between this system and the current standard was only 14.79%. The combination of these two evaluation methods provides a new method for screening and cultivating “dual resistance” maize germplasm/varieties that possess both disease resistance and fumonisin production resistance.

The objective of this study is to screen Trichoderma strains which have inhibitory effect on the Pythium spp. causing maize stalk rot, and to clarify their taxonomic status, control efficacy and antifungal mechanism. This study will provide important resources for the research and development of biocontrol agent against Pythium stalk rot.

For the antagonistic strains screening, the inhibitory effect of Tichoderma strains on P. inflatum, P. arrhenomanes and P. aristosporum was tested by measuring the mycelia growth. The taxonomic status of Tr21 was determined by morphological and molecular characteristics. The effect of Tr21 on the mycelia morphology of Pythium spp. was observed in the laboratory. In order to analyze the effect of Tr21 fermentation broth on the membrane permeability of Pythium spp., propyridine bromide (PI) dye solution was used to stain, and the absorbance values of protein and nucleic acid in mycelia supernatant at different treatment times were detected. The effect of Tr21 fermentation broth on germination characteristics of maize seeds was tested by seed soaking with different concentrations of fermentation broth. The control efficacy of Tr21 on stalk rot was confirmed through greenhouse pot and field inoculation experiments.

From the 109 strains of Trichoderma spp., seven strains were screened with antagonistic activity against P. inflatum, P. arrhenomanes and P. aristosporum, and the inhibition rate was above 60%. The inhibition rate of Tr21 to three Pythium species reached 100%, the inhibition rate of 5×, 10× and 20× diluent to three Pythium species reached 100%, and the inhibition rate of 50× diluent to three Pythium species was also more than 55.56%. Tr21 strain was identified by morphological and molecular biology as T. afroharzianum. The results of microscopic observation showed that the fermentation broth of Tr21 could cause mycelial malformations, such as rough mycelia, increased mycelial branching, shortened nodes, and overflow of mycelia contents. The result of PI fluorescence stain showed that the cell membrane of three Pythium species was damaged by Tr21 fermentation broth, and the PI dye was more likely to penetrate the damaged cell membrane into the mycelium and stain the mycelia red. The results of nucleic acid and protein leakage showed that the absorbance values of the mycelia treated by the fermentation broth changed greatly. After treatment for 5 h, the OD₂₆₀ increased by 0.08 and OD₂₈₀ increased by 0.10, 0.11 and 0.10, respectively, indicating that the membrane of the mycelia was damaged, leading to the overflow of mycelia contents. The different concentrations of Tr21 fermentation broth had no effect on the germination characteristics of maize seeds, and the 20× diluent had the best effect on germination and growth of seeds. The results of pot experiment showed that 5× diluted fermentation broth of Tr21 had the best control efficacy on Pythium stalk rot caused by three Pythium species, which was 60.67%, 63.15% and 59.66%, respectively. The control efficacy on Pythium stalk rot of 5× diluent was the highest, reaching 82.25%, with a mass ratio of 1:100 (5× diluent to seed).

An effective T. afroharzianum strain Tr21 was obtained for preventing and controlling of maize Pythium stalk rot. The fermentation broth of Tr21 can lead to mycelia malformation, breakage, cell membrane damage and contents leakage, etc. In conclusion, the T. afroharzianum strain Tr21 is a promising biocontrol microbial.

This study aimed to establish a standardized and easy-to-operate grading criterion for maize root rot severity based on the aboveground symptoms at the seedling stage, and to clarify the effects of different disease severities on maize growth and yield.

The maize was planted with hole-sowing, and the inoculum of Fusarium graminearum was placed between the seeds of two holes for root rot inoculation. After emergence, the symptoms of root rot were observed continuously, and the grading criterion of disease severity was established based on the symptoms of the aboveground parts at the V5 stage. The agronomic traits of plant height, stem base diameter, stem diameter, and ear height of plants with different grades were measured at the V11 stage and dough stage, respectively. At the same time, the incidence of stalk rot, which developed from root rot, was investigated at the dough stage. After harvesting, yield-related traits, including ear diameter, ear length, bare top length, row number per ear, kernel number per row, 100-kernel weight, and kernel weight per ear, were measured. Then, the relationship between root rot severity and yield-related traits was analyzed.

At the V1 stage, the symptoms of yellowing leaf and wilting tip, seminal root and mesocotyl with yellowish-brown lesions, and some mesocotyl with browned vascular tissue were observed. At the V3 stage, the mesocotyl and root system of the diseased plants were necrotic to varying degrees, and the aboveground part of some plants was dead due to the necrotic mesocotyl. At the V5 stage, it can be classified into the following three types, i.e., leaves showed yellowing to varying degrees, or even gradually withered; the plant appearance was a dwarf and developed slowly and stagnantly; the plant died. Furthermore, the severity of root rot was divided into grades 1, 3, 5, 7 and 9 based on the aboveground symptoms at the V5 stage. Investigation of agronomic traits showed that plant height, stem base diameter, stem diameter and ear height decreased gradually with the increase of disease grade of root rot at the V11 stage and dough stage. At the same time, the development of most 9-grade plants was inhibited or stagnated, and showed tillering or deformity. The investigation of stalk rot indicated that some of the root rot plants developed into stalk rot in the late growth stage. The incidence of stalk rot showed a decreasing trend with the increase of root rot grade. The analysis of yield traits showed that the grade of root rot disease was negatively correlated with seven yield-related traits. It could cause more than 50% of the yield loss at the 5-grade, and even no grain at the 9-grade.

Based on the observation and description of the symptoms of maize root rot, an efficient and easy-to-operate grading criterion reflecting disease severity was formulated according to the aboveground symptoms at the V5 stage. Root rot disease can reduce the plant height, stem diameter and ear height, and easily lead to cause stalk rot. The severity of root rot is negatively correlated with yield-related traits. The results of this study are expected to guide resistance evaluation of varieties against root rot, disease control and yield loss assessment.

The objective of this study is to establish a rapid detection method for Puccinia polysora during the incubation period based on the nested PCR, and to provide support for prediction and control of southern corn rust.

The nested PCR primers for the specific detection of P. polysora were designed using the variant region of the ITS sequence, including outer primer NX471-F/ITS4 and inner primer NX255-F/NX255-R. For amplification, the 20 µL PCR reaction mixture contained: Ex Taq DNA polymerase (5 U·μL^-1) 0.15 μL, 10×Ex Taq Buffer (Mg²⁺ plus) 2 μL, dNTP Mixture (2.5 mmol·L^-1 each) 1.6 μL, forward and reverse primers (10 μmol·L^-1) 0.3 μL each, template DNA 1 μL, ddH₂O 14.65 μL. The PCR program was performed as follows: the outer primer NX471-F/ITS4 was used for the first amplification and denaturation at 95℃ for 7 min, 30 cycles of denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 45 s, and a final extension at 72℃ for 7 min. The product was diluted 20 times and used as the template for the second run of the nested PCR, which was amplified with the inner primer NX255-F/NX255-R, and the PCR program was denaturation at 95℃ for 7 min; 30 cycles of 95℃ for 30 s, 66℃ for 30 s, and 72℃ for 26 s; 72℃ for 7 min. At the same time, the inner primer was selected for conventional PCR, in which the reaction mixture was the same as the nested PCR, and the amplification conditions were the same as the second run, except that the number of reaction cycles was 38. Under these conditions, the specificity was detected for P. polysora, Puccinia sorghi, Melampsora laricipopulina and seven other common maize pathogens, and the sensitivity of nested PCR was tested using genomic DNA of P. polysora and DNA from artificially inoculated leaves.

Nested PCR could specifically detect P. polysora from all tested fungi, with a 255 bp target fragment. The lowest detection limit of the nested PCR was 10 fg·μL^-1, and the sensitivity was 500 times that of the conventional PCR. For the samples containing 500-2.5×10⁴ urediniospores per gram of artificially inoculated leaves, the detection rates of conventional PCR and nested PCR were 0 and 85.71%, respectively. Nested PCR could detect at least 1 000 urediniospores. For the samples containing 1-7 uredia per gram of artificially inoculated leaves, the detection rates of conventional PCR and nested PCR were 76.19% and 100%, respectively. Conventional PCR and nested PCR could detect at least 2 and 1 uredia, respectively. For the samples containing 1-7 infection sites per gram of artificially inoculated leaves, the detection rates of conventional PCR and nested PCR were 14.29% and 66.67%, respectively. Conventional PCR and nested PCR could detect at least 6 and 3 infection sites, respectively.

With NX471-F/ITS4 as the outer primer and NX255-F/NX255-R as the inner primer, a detection method for P. polysora was established based on the nested PCR, which could quickly, efficiently and accurately detect the P. polysora during the incubation period in maize leaves.