Note: Effect of ear bagging systems on<Emphasis Type="Italic">Aspergillus flavus</Emphasis> kernel infection and aflatoxin contamination of corn hybrids grown in the field

Field studies were conducted for 4 years to determine the effect of various ear bagging systems onAspergillus flavus kernel infection and aflatoxin production in developing ears of corn hybrids. Each year, corn hybrids were grown on a Myatt loam (low water-holding capacity) and a Leeper silty clay loam (high water-holding capacity). Corn ears were inoculated withA. flavus using the side-needle technique 7 days after midsilk (50% of the plants in the plot had silks emerged). For the first 2 years, inoculated ears were covered with either white or black paper pollination bags at approximately 14 days after inoculation. During the last 2 years, inoculated ears were covered with either a brown paper pollination bag or a clear plastic zip-lock bag. Daily maximum temperatures were increased 2 to 4°C in all of the bagging systems over ambient temperatures. The bagging systems had a limited effect on aflatoxin production andA. flavus kernel infection in the Leeper silty clay loam. In the Myatt loam, ears covered with plastic bags had higher levels of aflatoxin contamination andA. flavus kernel infection compared with ears covered with paper bags. http://www.phytoparasitica.org posting May 4, 2007.     

Comparison of different inoculating methods to evaluate the pathogenicity and virulence of Aspergillus niger on two maize hybrids

A two-year field study was conducted to determine the effects of artificial inoculation techniques on the pathogenicity and virulence of Aspergillus niger kernel infection on two maize hybrids. Test plants included in the study were hybrids resistant and susceptible to Aspergillus flavus to determine if the host resistance mechanisms that limited A. flavus infection would also suppress A. niger infection. Ears were inoculated with the silk-channel, side-needle, and spray techniques 7?days after midsilk (50% of the plants in a plot had silk emerging). Ears were also inoculated with a modified-pinbar technique 21?days after midsilk. Kernel infection in 2008 in inoculated plants ranged from 2% to 11% and from 2% to 45% in the resistant and susceptible hybrids, respectively. In 2009, kernel infection in inoculated plants ranged from 13% to 32% and from 10% to 67% in the resistant and susceptible, respectively. The silk-channel, side-needle, and modified-pinbar techniques produced significantly higher levels of kernel infection in the susceptible hybrid in both years than the spray technique. When hybrids were compared, the silk-channel, side-needle, and modified-pinbar techniques induced significantly higher levels of infections in the susceptible hybrid than in the resistant hybrid in 2008 and 2009. The level of A. niger pathogenicity and virulence increased when conidia were placed inside the husks of developing ears by wounding (modified-pinbar and side-needle techniques) or non-wounding (silk-channel technique) inoculation methods. Although A. niger kernel infection was significantly lower in the A. flavus resistant hybrid compared to the A. flavus susceptible hybrid, A. niger infection levels were much higher than A. flavus infection levels typically observed in both of these hybrids in past studies.     

Systemic infection of sorghum and corn by conidia ofSclerospora Sorghi

Conidia ofSclerospora sorghi, obtained from either systemically-infected or local-lesion-infected leaves of sorghum (cv. Vidan), were capable of inducing typical downy mildew systemic infection, including oospore formation, in sorghum and corn hybrids. Very young inoculated seedlings displayed chlorotic systemic symptoms already on the first leaf, and often died at fourth-leaf stage. Systemic infection was induced by conidia on sorghum 1–14 days old at inoculation. Incidence of infection was much higher and symptoms less delayed when the shoot rather than coleorhizas of young sorghum and corn seedlings were inoculated; in two-week-old sorghum with three leaves, inoculation of the coleoptile or of the base of the second and third blades resulted in systemic infection; with coleoptile inoculation partial leaf chlorosis was delayed until the fourth-or fifth-leaf stage, showing that penetration without symptoms had occurred as far as the meristematic tissues of young leaves still within the leaf tube. Conidial inoculation of young sorghum tillers sprouting after cutting down healthy mother shoots resulted in systemic infection. Conidial inoculum is deemed to be the probable major means for systemic infection of corn and sorghum sown in fields in which oospores are not present; inoculation of new tillers of forage sorghum by conidia from infected plants in a neighboring field can explain the rise in numbers of plants systemically stricken. Two sweet corn hybrids — one considered resistant in the field, the other very susceptible — proved equally susceptible when inoculated with conidia at 5 days of age.     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

Period of susceptibility of almonds to aflatoxin contamination during development in the orchard

Almonds can be contaminated with aflatoxins, produced mainly by Aspergillus flavus and A. parasiticus. Infection can be facilitated by insect injuries during hull split, which begins four to six weeks before harvest. Within this period, it is unknown which kernel stages are most susceptible to aflatoxin contamination. Developing almonds of the Nonpareil cultivar were inoculated weekly with a spore suspension of A. flavus or A. parasiticus for five weeks after hull split in 2013. The almonds were infested with eggs of the lepidopteron navel orangeworm (NOW) (Amyelois transitella) before each spore inoculation. Aflatoxin levels were quantified at harvest using HPLC. Aflatoxin contamination was consistently higher in NOW-damaged kernels, although aflatoxins were also detected in undamaged kernels at each inoculation date. Insect injury is not required for kernel infection but it is a key risk factor for high aflatoxin contamination. Laboratory inoculations were also performed on Nonpareil almond kernels collected during the summers of 2013 and 2015. Aflatoxin levels were significantly lower on dried almonds but the ability to produce aflatoxins was restored when almonds were incubated with high humidity or when the Aspergillus species were inoculated on almond meal agar amended with ground kernels. Therefore, aflatoxins can accumulate in kernels with low a_w, should sufficient moisture favors aflatoxin production. In our field experiment, the orchard micro-climate had sufficient humidity to enable aflatoxin production in both damaged and undamaged dried kernels.     

Evaluating commercial maize hybrids for resistance to gibberella ear rot

An integral component of breeding maize for resistance to Fusarium graminearum ear rot is the identification of resistant genotypes. Since natural infection is not consistent from year to year, maize researchers must use manual techniques to inoculate the plant material with fungal spores. Information is presented here on site resistance of commercial maize hybrids to F. graminearum over three years and at two locations. Additionally, results of an investigation on the two predominant techniques of inoculating maize, the silk channel and kernel inoculation methods, are reported. Of 61 commercial hybrids tested, only two were ranked as moderately resistant to the fungus by both inoculation methods. These two hybrids also had a stable response to the F. graminearum infection across seven environments when the silk channel inoculation method was used. The majority of the hybrids were ranked as either susceptible or highly susceptible and less than 10% of the hybrids had a stable response to fungal infection. In the investigation of methodology, it was concluded that silk browning would be the least laborious way to identify the ideal time to complete silk channel inoculations. It was found that kernel inoculations using the pin inoculation method should take place between 11 and 15 days after 50% silking to achieve proper hybrid discrimination. Mist irrigation increased mold severity ratings and resulted in greater discrimination between hybrids with varying levels of resistance to F. graminearum infection.     

Advances in the Development of Host Resistance in Corn to Aflatoxin Contamination by Aspergillus flavus

ABSTRACT Aflatoxins are toxic, highly carcinogenic secondary metabolites of Aspergillus flavus and A. parasiticus, which when produced during fungal infection of a susceptible crop in the field or after harvest contaminate food and feed and threaten human and animal health. Although there are several management strategies that may reduce aflatoxin contamination of corn, the preeminent strategy for elimination of aflatoxin is to develop preharvest host resistance to aflatoxin accumulation. This strategy has gained even greater prominence due to recent discoveries of natural resistance in corn that can be exploited in plant-breeding strategies. The ability to identify resistant corn genotypes has been enhanced by the development of a laboratory kernel-screening assay and by a strain of A. flavus genetically engineered to produce beta-glucuronidase, an enzyme whose activity can be monitored to assess the degree of fungal infection in kernels. Investigations of resistant corn genotypes have associated kernel pericarp wax characteristics with resistance, identified kernel proteins associated with resistance to and inhibition of fungal growth or aflatoxin biosynthesis, and identified chromosome regions associated with resistance to Aspergillus ear rot and aflatoxin production. Such research advances could lead, in the near future, to commercially available, agronomically acceptable corn lines with multiple preharvest resistances to aflatoxin contamination.     

Susceptibility of pure and hybrid willows to isolates ofMelampsora epitea rust

Pure species and F₁ hybrid families ofSalix viminalis andS. dasyclados were tested for resistance to four single uredinium isolates ofMelampsora rust in laboratory experiments using excised leaves. Rust isolates were derived from:S. viminalis, S. dasyclados, aS. viminalis x triandra hybrid, andS. daphnoides. Incidence of infection, number of uredinia per leaf, and numbers of spores per uredinium were measured. As expected, the isolate fromS. daphnoides did not infect any of the willow species or hybrids tested. For the other three rust isolates that were tested, the parent from which the isolate was derived was susceptible, the other parent was resistant, and hybrids were intermediate in resistance for incidence and uredinia per leaf. These patterns indicate additive inheritance of these resistance traits in hybrids. Numbers of spores per uredinium were similar on the hybrids and the susceptible parent for one rust isolate, suggesting dominant inheritance of this trait in the hybrids.     

Induction of peroxidase as a disease resistance response in resistant (<Emphasis Type="Italic">Hibiscus trionum</Emphasis>) and susceptible (<Emphasis Type="Italic">Althea armeniaca</Emphasis>) species in the family malvaceae

The pathogen-induced biosynthesis of peroxidase (PO) in resistant and susceptible wild species of Malvaceae was studied. Specifically,Hibiscus trionum was found to be resistant to the wilt pathogenVerticillium dahliae, andAlthea armeniaca was susceptible. After inoculation withV. dahliae, PO activity increased more rapidly in the resistant plant: increased activity inH. trionum was noted within 1 h after inoculation, remained high for 5 h, and decreased after 18 h. After 5 days, the levels did not differ from the control. In the susceptibleA. armeniaca, PO activity did not differ statistically from the control even after 18 h, but was significantly higher after 5 days. Electrophoretic analysis of inoculated tissue extracts showed the occurrence of two new isoforms inh. trionum after inoculation; new isoforms did not occur inA. armeniaca. A chitin affinity chromatography column showed the presence of a chitin-binding peroxidase isozyme in healthyH. trionum that increased dramatically after inoculation. Two chitin-binding peroxidase isozymes were observed in healthyA. armeniaca: one (R _f 0.26) did not increase and the other (R _f 0.89) evinced only a small increase after inoculation.     

Antioxidant,ethylene and membrane leakage responses to powdery mildew infection of near-isogenic barley lines with various types of resistance

Leaves of powdery mildew-susceptible barley (Hordeum vulgare cv. Ingrid) and related near-isogenic lines bearing various resistance genes (Mla12, Mlg or mlo5) were inoculated with Blumeria graminis f. sp. hordei race A6. Fungal attack induced several-fold increases in ethylene emission and electrolyte leakage in leaves of susceptible Ingrid beginning 3 days after inoculation. Activities of peroxidase, superoxide dismutase, glutathione S-transferase, ascorbate peroxidase and glutathione reductase enzymes were induced markedly in susceptible leaves 5–7 days after inoculation. Similar, but less pronounced pathogen-induced changes were detected in inoculated leaves of Mla-type resistant plants that show hypersensitive cell death upon inoculation, and, to an even lesser extent, in the Mlg and mlo lines, where no visible symptoms accompanied the incompatible interaction. Glutathione content increased only in susceptible barley 7 days after inoculation. Catalase activity, total ascorbate content and redox state were not influenced by inoculation in any of the genotypes. The activity of dehydroascorbate reductase was significantly reduced 3–5 days after inoculation in the susceptible parental plants and after 5 days in Mla and Mlg lines, while it was stable in the mlo barley. Slightly elevated levels of H₂O₂ were observed in the inoculated resistant plants. In contrast, H₂O₂ content decreased in the susceptible line 7 days after pathogen attack. These data indicate that high levels of antioxidants are involved in the compatible interaction of susceptible barley and powdery mildew by protecting the pathogen from oxidative damage.     

A recessive resistance gene of alfalfa to alfalfa mosaic virus and impact of high ambient temperature on virus resistance

Twenty plants of alfalfa cv. Beaver were screened for resistance to alfalfa mosaic virus (AIMV) severe strain A-515. ELISA screening on both inoculated and apical leaves at fixed temperature (20°C day, 16°C night) suggested the following three types of clonal response to AIMV infection: extremely resistant, AIMV not normally detected from either inoculated or apical leaves; resistant, AIMV detected from inoculated leaves only and did not spread systemically; susceptible, AIMV detected from both inoculated and apical leaves. When plants in the second category were maintained at high temperature (30°C), AIMV was detected from inoculated and apical leaves 6 and 12 days after inoculation, but was not detectable in the apical leaves thereafter. Plants in the first category remained extremely resistant at all temperatures tested. The results of comparative tests using progeny plants of extremely resistant, resistant and susceptible plants, and of their hybrids, suggested that the resistance to AIMV A-515 was controlled by a temperature-dependent recessive gene.     

Relationships between aflatoxin production and sclerotia formation among isolates of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> from the Mississippi Delta

Aspergillus section Flavi isolates, predominately A. flavus, from different crops and soils differed significantly in production of aflatoxin and sclerotia. About 50% of the isolates from corn, soil and peanut produced large sclerotia, while only 20% of the rice isolates produced large sclerotia. There was a higher frequency of small sclerotia-producing isolates from rice compared to the other sources and isolates that did not produce sclerotia were significantly less likely to be toxigenic than strains that produced large sclerotia.     

Adzuki bean leaf infection by <Emphasis Type="Italic">Phytophthora vignae</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> and resistance evaluation using detached leaves inoculated with zoospores

We examined the response of adzuki bean leaves to infection by Phytophthora vignae f. sp. adzukicola and determined whether inoculated leaves can be used to evaluate cultivar resistance. Detached adzuki bean leaves were inoculated with zoospores, and the resulting symptoms were diagnosed. Resistant reactions were characterized by dark brown, speckled lesions or a lack of symptoms, while susceptible reactions were characterized by water-soaked spreading lesions. In an inoculation experiment using a combination of three differential cultivars and three races, the response of 10-day-old primary leaves accurately differentiate between race-specific resistance and susceptibility of adzuki cultivars.     

Assessment of early blight (<Emphasis Type="Italic">Alternaria solani</Emphasis>) resistance in tomato using a droplet inoculation method

A droplet inoculation method was used for evaluation of tomato resistance to early blight, a destructive foliar disease of tomato caused by Alternaria solani (Ellis and Martin) Sorauer. In this test method, leaflets are inoculated with small droplets of a spore suspension in either water or a 0.1% agar solution. Early blight resistance was evaluated based on lesion size. The droplet method better discriminated the level of resistance (P < 0.001) for a range of spore densities in comparison with the more commonly used spray inoculation method. Lesions generated by droplet inoculation at 7 days after inoculation ranged from small flecks to almost complete blight with an exponential-like distribution of lesion sizes. Significant correlations (r = 0.52, 0.58, and 0.63, P < 0.001) were observed across three glasshouse tests of 54 accessions including wild species using the droplet method. The most resistant accessions included wild species: one accession of Solanum arcanum, three accessions of Solanum peruvianum, one accession of Solanum neorickii, and one of Solanum chilense. Solanum pennellii and Solanum pimpinellifolium accessions were susceptible, whereas Solanum habrochaites and Solanum lycopersicum accessions ranged from susceptible to moderately resistant. The droplet test method is simple to apply, offers a fine discrimination of early blight resistance levels, and allows objective evaluation.     

玉米品种抗茎腐病鉴定

１９９１￣１９９５年采用自然发病和土壤接种方法鉴定了１５５０份玉米品种对茎腐病的抗病性,表现高抗的３９７份,中抗３０４份,中感３６４份,高感４８５份,经重复鉴定,筛选出７６份抗性稳定的自交系和杂交种。不同抗性品种用肿囊腐霉菌和串珠镰刀菌接种测定,抗性反应基本一致,人工接种比自然发病鉴定效果好,通过病圃自然发病初筛,人工土壤接处复选,可准确鉴定大量材料的抗病性。     

Induction of systemic resistance to<Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis> in bean by a benzothiadiazole derivative and rhizobacteria

Plants have developed mechanisms to resist secondary infection upon inoculation with a necrotizing pathogen, chemical treatment as well as treatment with some non-pathogenic microorganisms such as rhizosphere bacteria. This phenomenon has been variously described as induced systemic resistance (ISR) or systemic acquired resistance. In the present study, the chemical benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH, acibenzolar-S-methyl), and the rhizobacteriaPseudomonas aeruginosa KMPCH andP. fluorescens WCS417 were tested for their ability to induce resistance toColletotrichum lindemuthianum in susceptible and moderately resistant bean plants (Phaseolus vulgaris L.). BTH induced local and systemic resistance when bean leaves were immersed in 10⁻³ to 10⁻⁷ M BTH 3 days before the challenge inoculation. At a high concentration (10⁻³ M), BTH induced resistance of the same order as resistance induced by the pathogenC. lindemuthianum, although at this high concentration BTH appeared to be phytotoxic. Soil and seed treatment with 1 mg kg⁻¹ BTH protected beans against anthracnose. BTH-mediated induced resistance was effective in susceptible and moderately resistant plants.P. aeruginosa KMPCH induced resistance in bean againstC. lindemuthianum only in a moderately resistant interaction. KMPCH-567, a salicylic acid mutant of KMPCH, failed to induce resistance, indicating that salicylic acid is important for KMPCH to induce resistance in the bean—C. lindemuthianum system.P.fluorescens WCS417 could induce resistance toC. lindemuthianum in a susceptible and in moderately resistant interactions. http://www.phytoparasitica.org posting Jan. 16, 2002.     

Germination induces accumulation of specific proteins and antifungal activities in corn kernels

ABSTRACT This study examined protein induction and accumulation during imbibition and germination of corn kernels, as well as antifungal activities of extracts from germinating kernels against Aspergillus flavus and Fusarium moniliforme. Genotypes studied included GT-MAS:gk and Mp420, which are resistant to A. flavus infection and aflatoxin accumulation, and Pioneer 3154 and Deltapine G-4666, which are susceptible to A. flavus infection and aflatoxin accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved five protein bands that were present at higher concentrations in germinated kernels than in nongerminated kernels. Western blot analyses revealed that one of these proteins reacted with the 22-kDa zeamatin antiserum, and a zeamatin-like protein accumulated to a higher concentration in germinated kernels. Two protein bands from dry kernels that reacted with ribosome-inactivating protein (RIP) antiserum were identified as the 32-kDa proRIP-like form and an 18-kDa peptide of the two peptides that form active RIP. However, in germinated kernels, two protein bands that reacted with RIP antiserum were identified as two RIP-like peptides with a molecular mass of approximately 18 and 9 kDa. Purified RIP and zeamatin from corn inhibited growth of A. flavus. Bioassays of germinated kernel extracts from all four genotypes exhibited antifungal activity against A. flavus and F. moniliforme, with extracts from the susceptible genotypes showing greater inhibition zones. This study provides evidence of protein induction in corn kernels during imbibition or the early stages of germination, and the induced proteins may be related to our previous findings of germination-associated resistance in the corn kernel, especially in the susceptible kernels.     

Analysis of Accumulation Patterns of <Emphasis Type="Italic">Barley yellow dwarf virus-PAV</Emphasis> (BYDV-PAV) in Two Resistant Wheat Lines

Barley yellow dwarf (BYD) is one of the main viral diseases of small-grain cereals. This disease, reported on numerous plant species of the Poaceae family, is caused by a complex of eight viral species including the species Barley Yellow Dwarf Virus-PAV (BYDV-PAV), frequently found in western Europe. Resistance sources against BYDV-PAV are scarce and only identified in perennial Triticineae. Some BYDV-resistant wheat lines have been obtained by introgressing these resistances into bread wheat germplasms. Genetic and biological characterization of the resulting lines has been undertaken. However, little information on the resistant behaviour of these lines during the early stages of the infection process is available. To evaluate the resistance of two genetically distinct resistant lines (Zhong ZH and TC14), 1740 young plantlets, belonging to susceptible reference hosts (barley cv. Express and wheat cv. Sunstar), Zhong ZH or TC14 wheat lines, were inoculated in controlled conditions with French BYDV-PAV isolates. The infection process was monitored during the first 21 days after inoculation (DAI) using a semi-quantitative ELISA. A standardized protocol including five successive samplings of leaves from all inoculated plants and the collection of plant roots at the end of the monitored period was carried out. This protocol enabled an assessment of the infection percentage and the evolution of the viral load in plants from the 7th DAI to the 21st DAI. Statistical analyses of the BYDV infection kinetics using raw ELISA data, a model of the time-dependent variation of the percentage of infected plants and the area under concentration progress curves (AUCPC) demonstrated that Zhong ZH and TC14 lines (1) reduce the development rate of the BYD disease during the first days of infection, (2) decrease the infection efficiency of BYDV-PAV isolates, in the leaves, from 98.7% for susceptible plant genotypes to 81.9% and 71.7% for Zhong ZH and TC14, respectively, (3) reduce the virus load in the leaves of infected plants and (4) are not spared from BYDV infection, as 95.1% of Zhong ZH and 90.2% of TC14 inoculated plants accumulated viral particles in roots and/or in leaves at 21 DAI. These results confirm the BYDV-partial resistant behaviour of both Zhong ZH and TC14 lines. The development rate of the disease was the single parameter that allowed the distinction between the two resistant sources present in the tested lines.     

Changes in soluble and cell wall-bound hydroxycinnamic and hydroxybenzoic acids in sugarcane cultivars inoculated with <Emphasis Type="Italic">Sporisorium scitamineum</Emphasis> sporidia

The accumulation of soluble and cell wall-bound phenolics in the sugarcane stems of young plants from highly resistant cv. My 5514 and susceptible cv. B 42231, inoculated or not inoculated with smut sporidia, was studied. The ratio of inoculated to uninoculated plants of some cell wall-bound phenolics, such as ferulic, caffeic, and syringic acids increased for the resistant cv. My 5514, whereas it was maintained more or less constantly for the susceptible cv. B 42231. The highest increase of this ratio in the resistant cv. My 5514 corresponded to both caffeic and syringic acids. This could result in a better capacity to cv. My 5514 for an increase in the frequency of bridges between lignin fragments through ester-ether linkages for reinforcing the cell wall and major resistance to the disease. This reinforcement of the cell wall could provide an effective barrier to pathogen entry and spread. Soluble sub-fractions of all phenolics detected showed non-stable patterns. Caffeic acid, that regulates phenylalanine ammonia-lyase activity in sugarcane, showed a significant decrease in its titre at 24 h in the resistant cultivar, principally in the free soluble fraction, whilst the susceptible cultivar enhanced it. We hypothesise that the pathway of hydroxybenzoic acids is only activated once the level of p-coumaric acid justifies the accumulation of hydroxycinnamic acids required for reinforcing the cell wall after inoculation.