Efficacy of Trematophoma lignicola Petrak as a Biological Control Agent for Amaranthus retroflexus L.

The pathogenicity of Trematophoma lignicola on Amaranthus retroflexus was increased when its conidia were formulated in oilseed rape emulsion. However, this formulation did not reduce the dew period required to allow conidial germination and infection of the host, nor did it protect the conidia from desiccation before the onset of dew. The fungus gave effective control of A. retroflexus plants up to the 4-true-leaf stage. Plants with 5 to 6-true-leaves were infected and their dry weight reduced, but plants with more than 6-true-leaves were unaffected. Plants grown in warm conditions (18-21°C/12-15°C, day/night) were more likely to be successfully controlled than those grown in the cold (10-12°C/7-8°C; day/night). Significant dry weight loss of A. retroflexus plants were achieved at application volumes down to 100 l ha -1 at 5 ×10 6 conidia ml -1 . More effective control was achieved by application of conidia of T. lignicola than mycelial or pycnidial applications.     

Investigation of Spray Application of Microbial Herbicides Using Alternaria alternata on Amaranthus retroflexus

Inclusion of Alternaria alternata conidia in a spray formulation affected the distribution pattern on the target. The dry weight of Amaranthus retroflexus plants was reduced by more than 83% when A. alternata conidia (107 ml -1 ) were applied at 200 L ha -1 or greater and when given a 24 h dew period. At low application volumes (25 or 50 L ha -1 ) plant dry weight was reduced by only 29 or 54%. After 7-8 h dew period, conidial germination on the leaf surface was 11-19%. This increased to 62-91% after 24 h dew period. Counts of conidia on leaves indicated that up to 86% of the conidia sprayed were not retained on the target plant, or did not reach it. This is reflected in lesion numbers per unit area being only 3-5% of the calculated theoretical numbers. The results cast doubt on the suitability of A. alternata as a microbial herbicide for the control of Am. retroflexus .     

Trematophoma lignicola (Petrak): A Potential Microbial Herbicide for Control of Amaranthus retroflexus (L.)

Trematophoma lignicola (Petrak), sprayed on leaves at 106 conidia ml -1 in oil emulsion, kills Amaranthus retroflexus plants after a 16 h dew period. Application as a soil drench kills seedlings of this species.     

Outdoor Studies of the Efficacy of Alternaria alternata in Controlling Amaranthus retroflexus

Application of 250 L ha -1 containing 10 7 Alternaria alternata conidia ml -1 caused 61% reduction in dry weight and a 45% mortality in Amaranthus retroflexus plants growing in a wheat crop. However, only 10-22% of the applied conidia were retained on the leaf. In one experiment, conidia remained viable for 4 days on the leaf surface until conditions were favourable for germination. Competition from the wheat crop, as well as its creation of a moist micro-climate, improved the control of Am. retroflexus by A. alternata . Total control of Am. retroflexus may be difficult to achieve with A. alternata , but suppression to reduce or eliminate its competitive effect on crops is possible.     

Effects of Nutrition on Desiccation Tolerance and Virulence of Colletotrichum truncatum and Alternaria alternata Conidia

The promising mycoherbicides Colletotrichum truncatum and Alternaria alternata were grown respectively in liquid and solid semi-defined media. C. truncatum conidia produced in a medium with a C:N ratio of 5:1 showed higher desiccation tolerance (survival during storage) at 15% relative humidity and 25°C, greater germination on the host leaf and greater disease expression on Sesbania exaltata than those produced in media with C:N ratios of 15:1 or 40:1. Similar results were obtained with conidia of A. alternata produced on a medium with a C:N ratio of 15:1. Conidia washed with 0.9% (w/v) NaCl produced higher tolerance to desiccation, and greater disease incitement, than unwashed conidia of C. truncatum or conidia washed with water. In contrast, washing had no positive effect on desiccation tolerance in A. alternata .     

Factors Influencing the Efficacy of the Potential Microbial Herbicide Alternaria alternata (Fr.) Keissler on Amaranthus retroflexus (L.)

Alternaria alternata , applied to run-off at 107 conidia ml -1 and given a 6 h dew period, caused an 88.5% reduction in dry weight of 4 to 5 true-leaf plants of Amaranthus retroflexus . This level of control was achieved whether the inoculated plants were grown in the glasshouse or outside. Formulation of the pathogen in oilseed rape emulsion reduced its dew period requirement from 24 h or more to 8 h or less. For example, with 106 conidia ml -1 , equivalent effects were found with 8 h dew with emulsion formulation compared to 48 h dew with the Tween formulation. Allowing spray deposits to dry on inoculated plants for ca 7 h, before exposing to dew increased the efficacy of the pathogen.     

In vitro efficacy of some fungicides on mycelial growth of Alternaria alternata and Mucor pyriformis

Various chemical fungicides, systemic and non-systemic, were tested against fruit rot pathogens viz. Alternaria alternata and Mucor pyriformis for the evaluation of inhibition of mycelial growth. In A. alternata, among the systemic fungicides used, hexaconozole showed highest inhibition of mycelial growth followed by carbendazim and least effective was myclobutanil. While in M. pyriformis, hexaconozole showed highest inhibition and least effective was bitertanol. Among the non-systemic fungicides tested in both A. alternata and M. pyriformis, mancozeb showed highest inhibition of mycelial growth followed by capton and the least inhibition was shown by zineb.     

Studies of Spray Application of Microbial Herbicides in Relation to Conidial Propagule Content of Spray Droplets and Retention on Target

This paper addresses the hypothesis that the spray application system used will affect the retention of spray deposits of microbial herbicides on different plant structures. There was greater spray retention on the hypocotyl of 4 to 6-true leaf Amaranthus retroflexus plants (20-31%) sprayed with an aerosol-producing laboratory sprayer than with a hydraulic nozzle (3-5%). Spray deposition and retention from the hydraulic nozzle was increased by 28-42% when the nozzle was used horizontally. Spray droplets generally contained the expected conidial number for the conidial concentration but this was affected by increased inoculum density (size, concentration and mycelial contamination), causing a reduction in the actual number of conidia present, compared to the expected. Up to 40-50% of the conidia in the sprayed suspension could not be accounted for when conidium deposition was on a target. This loss could increase to 90-92% when the weed was sprayed under a crop canopy.     

Relationship between Fusarium graminearum and Alternaria alternata contamination and deoxynivalenol occurrence on Argentinian durum wheat

A mycological survey was carried out on durum wheat (Triticum durum) samples from the main production area of Argentina. The isolation frequency and relative density of species of dematiaceous fungi, and genus Fusarium were calculated. Alternaria alternata and Fusarium graminearum were the predominant fungal species. An analysis of deoxynivalenol (DON) natural contamination was also performed on a limited number of samples (60). DON contamination levels in positive samples ranged from 26 to 6400 μg/kg. The non-parametric techniques applied showed that there is a positive relationship between DON contamination and F. graminearum relative densities and a negative relationship between DON contamination and A. alternata relative densities. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of environmental factors on tenuazonic acid production by Alternaria alternata on soybean-based media

Aims: To determine the effects of water activity (a_W; 0·995–0·90), temperature (5, 18, 25 and 30°C), time of incubation (7–35 days) and their interactions on tenuazonic acid (TA) production on 2% soybean‐based agar by two Alternaria alternata strains isolated from soybean in Argentina. Methods and Results: TA production by two isolates of A. alternata was examined under interacting conditions of a_W, temperature and time of incubation on 2% soybean‐based agar. Maximum TA production was obtained for both strains at 0·98 a_W, but at 30 and 25°C for the strains for RC 21and RC 39, respectively. The toxin concentration varied considerably depending on a_W, temperature, incubation time and strain interactions. TA was produced over the temperature range from 5 to 30°C and a_W range from 0·92 to 0·995, however at 5 and 18°C little TA was produced at a_W below 0·94. Contour maps were developed from these data to identify areas where conditions indicate a significant risk for TA accumulation. Conclusions: The optimum and marginal conditions for TA production by A. alternata on soybean‐based agar were identified. The results indicated that TA production by A. alternata is favoured by different temperatures in different strains. Significance and Impact of the Study: Data obtained provide very useful information for predicting the possible risk factors for TA contamination of soybean as the a_W and temperature range used in this study simulate those occurring during grain ripening. The knowledge of TA production under marginal or sub‐optimal temperature and a_W conditions for growth are relevant as improper storage conditions accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality.     

Aflatoxins in sunflower seeds: Influence of Alternaria alternata on aflatoxin production by Aspergillus parasiticus

The aim of the present work was to determine the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus.A mixture of spores of both strains was inoculated in sunflower seeds at 0,90 a_w, and incubated for 42 days at 28 °C ±1.The cultures were observed and analyzed every 7 days to determine the infection level of the seeds and the production of aflatoxins. Results showed that when the seeds were inoculated only with Aspergillus parasiticus, 100% were infected from the 7th day.When Aspergillus parasiticus and Alternaria alternata were simultaneously inoculated the infection level of the seeds was 100% for Aspergillus parasiticus following 7 days of inoculation and 0% for Alternaria alternata. After the 14th day of inoculation there was no significant difference in the infection percentage of both strains (approximately 80% of each one). As far as toxin production is concerned a remarkable decrease was observed when seeds were inoculated with both strains simultaneously.In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds but would either degrade the aflatoxins by Aspergillus parasiticus or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.     

Use of a Novel Sporulation Monitor to Quantify the EVects of Formulation and Storage on Conidiation by Dried Mycelia of the Entomopathogenic Fungus Zoophthora radicans

A novel piece of equipment, the sporulation monitor, is described for the comparison of conidia production from mycelia receiving diVerent formulation and storage treatments. This equipment was used to compare the viability of Zoophthora radicans mycelial samples treated either with 10% maltose solution or with distilled water before drying and storage for 0-12 weeks at 4oC. Freshly dried maltose-treated mycelial mat samples produced significantly more conidia for a significantly longer time than distilled water-treated mat samples of the same age. Very few conidia were produced from mats in either treatment after storage for 4 weeks or longer. There were great diVerences in conidia production from mycelial mat samples produced in diVerent fermenter runs. These results are discussed in relation to the potential for the use of dried mycelia in biological control programmes.     

Mutations at the Asc locus of tomato confer resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M₂ mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A a. lycopersici are discussed.     

Production of phytotoxic spore germination liquids by Alternaria brassicae and A. brassicicola and their effect on species of the family Brassicaceae

Experiments assessed the susceptibility of Brassica spp. and non-Brassica spp. in the family Brassicaceae to infection by Alternaria brassicae and A. brassicicola, and determined the sensitivity of the host species to spore germination liquids (SGLs) produced by the pathogens on B. napus leaves. There was a wide range of sensitivity to the pathogens. Brassica spp. were generally more susceptible, and some non-Brassica spp. (Barbarea vulgaris and Capsella bursa-pastoris) were immune to A. brassicicola. Measurable damage was caused by SGLs but with significant variation between host species. Non-hosts and weak hosts also showed necrosis. It was concluded that, in the case of both pathogens, the toxic factors in these SGLs were host-selective. Selectivity in toxin production was also demonstrated in relation to the host surface or growing medium in which spores germinated. A substantial amount of toxin was produced on all Brassicaceae tested but not on unrelated species (Triticum aestivum, Pisum sativum and Lycopersicon esculentum). Neither pathogen produced measurable amounts of toxin when cultured in Czapek (Dox) broth.     

Effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments were done under controlled environment and glasshouse conditions to study the effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brussicae) on oilseed rape (Brassica napus). On leaves of potted oilseed rape plants (cv. Bienvenu) inoculated with A. brassicae conidial suspensions, the severity (number of lesions cm^-2) of dark leaf spot increased as inoculum concentration increased from 80 to 660 spores ml^-1and as leaf age increased from 4 to 14 days. On pods on detached racemes of spring oilseed rape (cv. Starlight), the incidence of dark pod spot (% of pods diseased) increased as inoculum concentration increased from 80 to 10⁴spores ml^-1. Increasing inoculum concentration above 10⁴spores ml^-1did not increase the incidence but did increase the severity of dark pod spot. A minimum wetness period of 4 h was needed for infection of oilseed rape leaves (cv. Envol) by A. brussicue at 18°C and disease severity increased with increasing wetness period up to 12 h. The length of dry interruptions after 3–8 h of initial wetness affected the severity of dark leaf spot. A second wetness period increased the severity of dark leaf spot if the dry interruption was ≤ 6 h and if the first wetness period was ≤ 8 h. The incubation period of A. brassicae decreased from 3.5 to 2.5 days as inoculum concentration increased from 80 to 660 spores ml^-on leaves (cv. Bienvenu) at 17–25°C and from 3.8 to 1.0 day as inoculum concentration increased from 80 to ≥2 ≥ 10³spores ml^-1on pods (cv. Starlight) at 18°C.     

Analysis of intraspecific and interspecific variation in the genus Alternaria by the use of RAPD-PCR

The RAPD-PCR profiles of 13 phytopathogenic Alternaria species and two closely related outgroups were examined using six different primers. Each species produced a distinct pattern of DNA fragments which were used as a measure of the degree of relatedness between species. A. brassicae isolates of diverse origin showed high levels of similarity but little similarity was noted between other species. The closest interspecific genetic distances were recorded between A. citri, A. alternata and A. longipes. The outgroup genera Embellisia and Stemphylium, which are recognised as distinct, could not be clearly separated using RAPD banding criteria, suggesting a high level of genetic diversity amongst these groups of fungi.     

Isolation and characterization of microsatellite markers from the phytopathogenic fungus Alternaria dauci

Eleven polymorphic microsatellite markers were isolated from the necrotrophic phytopathogenic fungus Alternaria dauci based on enriched genomic libraries. In order to assess allelic variability, the microsatellite loci were analysed in a collection of 43 isolates. The number of detected alleles in 11 loci ranged from two to 24 (mean 10.4). Test of cross-species amplification and sequencing of the resulting amplicons showed that some of these microsatellites could be used in different species such as Alternaria solani, Alternaria bataticola and Alternaria zinniae.     

Effects of Temperature, Humidity and Inoculation Method on Susceptibility of Schistocerca gregaria to Metarhizium flavoviride

The effects of temperature on conidial germination and susceptibility of adults of the desert locust , Schistocerca gregaria, to four isolates of Metarhizium flavoviride were determined . In addition , the effects of inoculation method (topical or spray) , spore carrier (oil or water) and ambient relative humidity (RH) on susceptibility of the locust to the most thermotolerant isolate (Mf324) were investigated . There were differences among the isolates in the effects of temperature on germination of conidia after a 24 - h incubation period . Over 90% of conidia of all isolates germinated after 24 h at 30 o C . In contrast , at 40 o C , none of the isolates germinated for up to 72 h . However , there were differences in germination between the isolates at 35 o C . Locust mortality and disease progression were significantly affected by temperature . At both 25 and 30 o C , all isolates induced 98 - 100% mortality within 8 days; however , there were differences between isolates at 35 o C . None of the isolates caused significant mortality at 40 o C . Humidity and inoculation method had no influence on levels of mortaility in fungus treated locusts . In contrast , carrier type significantly affected cumulative mortality . Topical oil treatment resulted in higher overall mycosis than the three other treatments . Control mortality on the other hand was strongly affected by inoculation method and to a lesser extent by humidity and carrier . In these conditions , application of oil by spray was generally toxic at all humidities whereas topical application of water was most toxic at near saturation . The results of these laboratory studies demonstrate the importance of strain selection , formulation and application method in the development of a microbial control agent against the desert locust . Low RH should not impede use of this fungus under dry conditions .