Screening maize genotypes for resistance to Fusarium verticillioides comparing in vivo plant response and in vitro bioassays

--Balconi, C, Lupotto, E, Lanzanova, C, Conti, E, Gualdi, L, Mazzoleni, A, Valoti, P, Motto, M

The availability of reliable methods for the screening and evaluation of maize genotypes for improving tolerance to Fusarium attacks is an invaluable tool in breeding programmes to increase crop protection against fungal infection. Numerous Fusarium species are widespread pathogens in cereals. They infect small grain cereals (wheats, barley, oats, rice, triticale, sorghum, millet) and maize (mostly F. verticillioides), causing root, stem, and ear rot diseases in both temperate and semitropical areas of the world. Some Fusarium strains also produce mycotoxins that can be formed in infected plants before harvesting, or in grains during post-harvest storage. The occurrence of mycotoxins in cereal grains is a great concern worldwide, because their presence in feed and foods is often associated with chronic or acute mycotoxicoses in livestock and also in humans. In this respect, the development of improved maize genotypes with increased resistance to F. verticillioides using breeding and biotechnology strategies is important. The aim of our research is to develop a rapid and reliable screening method to evaluate maize genotypes for resistance to the infection of this pathogen.

For this purpose, six inbred lines released by the Maize Section of Bergamo (Lo904, Lo1010, Lo1096, Lo1067, Lo1095, Lo1124) (Bertolini et al., Maydica 45:73–87, 2000) were used as the experimental material. The inbreds were tested in field experiments with Silk Channel Inoculation Assay (SCIA) on adult plants, and by in vitro bioassays with Kernel Inoculation Assay (KIA) to follow the progression of F. verticillioides infection in inoculated maize germinating seeds. The results of the in vivo and in vitro experiments were compared i) to evaluate if the in vitro bioassays reflect in vivo plant response to Fusarium attack, and ii) to offer a rapid screening test for genotype evaluation.

For both SCIA and KIA protocols F. verticillioides was grown on PDA plates at 26 C until the mycelium covered the surface of the plate, and used for fresh spore inoculum production. For the field experiments, plants were hand-pollinated and SCIA applied at two different stages of kernel development at 3 and 6 days after pollination (DAP); controls were non-inoculated and sterile water-inoculated plants. According to previous information (see Reid, Hamilton, and Mather, Technical Bulletin 1996-5E, research branch, Agriculture and Agri-Food Canada, 1996, method for F. graminearum), amounts of 2 ml, containing two spore concentrations of 10⁵ and 5×10⁵/ml, were employed for the SCIA tests. Inoculation was performed by spore injections into the silk channel of each primary ear. Ears were manually harvested, hand de-husked; the severity of ear rot symptoms was evaluated using rating scales based on the percentage of kernels with visible symptoms of infection, such as rot and mycelium growth. As reported by Reid et al. (1996), the visual rating scale consists of 7 classes based on percentage of visibly infected kernels (Disease Severity Rating-DSR-: 1=0%-no infection; 2=1–3%; 3=4–10%; 4=11–25%; 5=26–50%, 6=51–75%; 7=76–100%). Individual ear ratings using a visual scale, as described above, allowed a discernible screening of the six genotypes tested for Fusarium resistance. The kernel developmental stage at inoculation was discovered to be an important technical parameter: for all the genotypes tested, inoculations gave the best results when applied at 3 DAP. At this stage silks are elongated and green. Disease severity scores were lower for ears of the same genotypes infected at 6 DAP: at this latter stage silks appear wilted and began silk senescence, a physiological change apparently altering the suitability of silk for permitting the growth of ear-rotting organisms. The spore concentration of 10⁵/ml, applied for inoculation at 3DAP, gave maximum differentiation among genotypes, ranging from the most resistant Lo904 (DSR: 2.58 ± 0.49), to the most susceptible Lo1124 (DSR: 6.42 ± 0.66). Together with Lo904, Lo1010 and Lo1096 appeared to be more resistant (DSR-range: 2.58–3.89) than Lo1124, Lo1067 and Lo1095 (DSR-range:5.7–6.42). For all genotypes, the non-inoculated and sterile water-inoculated plants, as controls, showed no infection, with disease severity ratings around 1.

To obtain the optimal conditions for the in vitro KIA bioassays, sterile kernels were inoculated with a conidial suspension of the fungus by adding 125, 1250 or 12500 spores/seed. Inoculated and control kernels (sterile water inoculated) were allowed to continue germination for 7 days. For each genotype, records of the percent of maize kernels with visible fungus colonization and an “infection score”, were registered. The “infection score” rating scale consists of 6 classes related to kernel surface infection extent (Infection Score-IS-: 0=0% infected seed surface; 1=5–10%; 2=10–30%; 3=30–50%; 4=50–75%, 5=75–100%; 6=100% heavily infected). The progression of the infection on the developing vegetative tissues (radicle, coleoptile) was also recorded. Results indicated that an inoculum of 125 spores/seed was optimal to discriminate genotype response by the in vitro KIA bioassay. All Lo inbred lines tested showed a high percentage of seed colonized, but variability was observed for the extent of infection during the 7 days after inoculation. Lo904 and Lo1096 were resistant for all periods tested (IS-range: 1.14–1.75). On the other hand, Lo1095 and Lo1067 appeared significantly more susceptible 3 days after inoculation (IS-range: 2.33–4.58), and showed a very heavy contamination 7 days after inoculation (IS-range: 4.76–5.88). Interestingly, for these four genotypes, the indications obtained by in vitro KIA bioassays reflect field observations of resistance/sensitivity to F.verticillioides. Lo1010 and Lo1124, under the bioassay conditions previously described, showed response to Fusarium attack with scores intermediate between the most resistant and the most susceptible genotypes. Experiments are in progress to evaluate if higher or lower spore concentrations applied to KIA may improve the degree of differentiation among genotype response.

In conclusion, the results suggest that in vitro bioassays on mature kernels appear to be, after additional testing and standardization, a useful tool for screening and selfing maize plants with a superior level of protection against Fusarium verticillioides.

The work was developed within the framework of the Eu-funded project SAFEMAIZE (ICA4-CT2000-30033, Coordinator: Prof. Dave Berger, University of Pretoria, SA) in FP5.