The potential of silver nanoparticles to control Rhizoctonia solani (AG3-PT) growth in vitro

Silver nanoparticles (AgNPs) have known anti-microbial properties and therefore have the potential to be used to control fungal plant pathogens. In this study we investigated the growth of a plant pathogenic fungus, Rhizoctonia solani (AG3-PT) in the presence of AgNPs. The effect of AgNPs at two different levels (20 and 50 mg L-1) on hyphal growth and sclerotium production and viability in R. solani was investigated. The results showed that at 20 mg AgNPs L-1 R. solani hyphal growth was reduced along with the production of sclerotia. The results indicate that AgNPs have the potential to control R. solani growth and subsequent development of plant disease symptoms.

Diversity within Aspergillus niger Clade and Description of a New Species: Aspergillus vinaceus sp. nov.

Diversity of species within Aspergillus niger clade, currently represented by A. niger sensu stricto and A. welwitshiae, was investigated combining three-locus gene sequences, Random Amplified Polymorphic DNA, secondary metabolites profile and morphology. Firstly, approximately 700 accessions belonging to this clade were investigated using calmodulin gene sequences. Based on these sequences, eight haplotypes were clearly identified as A. niger (n = 247) and 17 as A. welwitschiae (n = 403). However, calmodulin sequences did not provide definitive species identities for six haplotypes. To elucidate the taxonomic position of these haplotypes, two other loci, part of the beta-tubulin gene and part of the RNA polymerase II gene, were sequenced and used to perform an analysis of Genealogical Concordance Phylogenetic Species Recognition. This analysis enabled the recognition of two new phylogenetic species. One of the new phylogenetic species showed morphological and chemical distinguishable features in comparison to the known species A. welwitschiae and A. niger. This species is illustrated and described as Aspergillus vinaceus sp. nov. In contrast to A. niger and A. welwitschiae, A. vinaceus strains produced asperazine, but none of them were found to produce ochratoxin A and/or fumonisins. Sclerotium production on laboratory media, which does not occur in strains of A. niger and A. welwitschiae, and strictly sclerotium-associated secondary metabolites (14-Epi-hydroxy-10,23-dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydro-21-oxo-aflavinine) were found in A. vinaceus. The strain type of A. vinaceus sp. nov. is ITAL 47,456 (T) (=IBT 35556).

Expression of the Histidine Kinase Gene Sshk Correlates with Dimethachlone Resistance in Sclerotinia sclerotiorum

Histidine kinases (HK) are implicated in virulence, vegetative mycelial growth, and osmotic and oxidative responses in pathogenic fungi. Our previous work showed that transcriptional levels of the group III HK gene Sshk are higher in field dimethachlone-resistant isolates of Sclerotinia sclerotiorum compared with sensitive isolates. However, it is not clear whether the overexpression of Sshk is the major mechanism for resistance to dimethachlone. In this study, we constructed Sshk silencing and overexpression vectors and assessed dimethachlone resistance levels, virulence, mycelial growth, and sensitivity to osmotic stress for the Sshk-silenced and -overexpression transformants. Overexpression of Sshk resulted in resistance to dimethachlone and increased sensitivity to various stresses and to the cell-wall-perturbing agents sodium dodecyl sulfate (SDS) and Congo red (CR). Compared with the parent isolate, Sshk-silenced transformants had reduced resistance to dimethachlone, significantly higher (P < 0.05) mycelial growth and virulence, and lower sclerotium production, and were less sensitive to various exogenous stresses such as sodium chloride. Compared with the parent sensitive isolate HLJMG1, dimethachlone resistance ratios of the three overexpression transformants ∆C101, ∆C21, and ∆C10 increased 168.1-, 189.5-, and 221.2-fold, respectively. The three overexpression transformants were more sensitive to CR and SDS than their parent isolate. These findings suggest that overexpression of Sshk is a major mechanism for dimethachlone resistance in some isolates of S. sclerotiorum, and that Sshk plays an important role in maintaining the integrity of the cell wall. Our findings reveal a novel molecular mechanism for dimethachlone resistance in plant-pathogenic fungi.

Characterization of Aspergillus section Flavi from pistachio soils in Iran

During 2012, soil samples from commercial pistachio orchards in three major production regions include Rafsanjan (Kerman Province, center of Iran), Damghan (Semnan Province, north-central Iran) and Feyz-Abad (Khorasan-e Razavi Province, northeastern Iran), were assayed on Dichloran Rose-Bengal Chloramphenicol agar (DRBC) and Aspergillus flavus-parasiticus agar media to quantify populations of Aspergillus species from the section Flavi. The mean propagule density of Aspergillus species from the Flavi section [log10 (CFU/g soil)] was higher in Feyz-Abad (3.06, 2.88–3.24), compared to Damghan (2.55, 2.44–2.65) and Rafsanjan (2.40, 2.26–2.54). A. flavus (69.7, 65.3 and 57.9%), A. parasiticus (19.6, 25.4, and 29.3%), and A. nomius (10.7, 9.3, and 12.8%) were the predominant species in the regions of Rafsanjan, Damghan, and Feyz-Abad, respectively. There were significant differences among sclerotia producing isolates of A. flavus in the sampling regions (p < 0.05). The percentage of sclerotium-producing isolates of A. flavus from Rafsanjan (14.5%) was much lower than Damghan (39.5%) and Feyz-Abad (41.4%). The A. flavus isolates from Damghan, Rafsanjan, and Feyz-Abad were toxigenic at 53.7%, 61.6%, and 60.4%, respectively. In Rafsanjan, aflatoxin B1 (AFB1), and AFB1 + AFB2 (aflatoxin B2) ranged from 274 to 553 ppb (393±17.11) and 394 to 3745 ppb, respectively, while AFB1, and AFB1 + AFB2 ranged from 257 to 392 ppb (285±13.18) and 415 to 1658 ppb, respectively, in Damghan. We found 16 and 20 vegetative compatibility groups (VCGs) for 41 and 37 nit mutant producing isolates of A. flavus from Rafsanjan and Damghan, respectively. From Damghan the VCG diversity for A. flavus isolates was greater (54%) than from Rafsanjan (39%). Because there were a few number of sclerotium-producing isolates of A. flavus, we did not determine the relationships between sclerotium production with VCGs and/or geographical distribution in the three pistachio production regions. This study was the first to determine the strain and VCG diversity of A. flavus soil isolates from Iranian pistachio orchards.

Sporogenic Effect of Polyunsaturated Fatty Acids on Development of Aspergillus spp

ABSTRACT Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans,Aspergillus flavus, and Aspergillus parasiticusis affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in someA. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intactveA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.

First Report of Sclerotium Production by Sclerotinia sclerotiorum in Soil on Infected Soybean Seeds

Stem rot of soybean caused by Sclerotinia sclerotiorum (Lib.) de Bary was not recognized as an important problem in the North Central Region of the United States until severe outbreaks occurred in 1992, 1994, and 1996 (2). Although sclerotia mixed with seeds are known to be important to the spread of this disease, the role of internally infested soybean seed in dissemination of the disease is unknown. Tu (1) demonstrated in dry bean, which differs from soybean in seed size and plant architecture, that internally infected seeds are important to the spread of the disease, by producing sclerotia in the soil after the seeds are planted. Experiments were conducted to determine if sclerotia are formed in soils from internally infected soybean seeds. Soybean seed from a field with 70% disease severity were collected and sorted into three classes: (i) normal quality seed, which included moderate or good seed; (ii) poor quality seed (shriveled and/or whitish); and (iii) seed of regular size with visible mycelial mats (S. sclerotiorum or Peronospora manshurica (Naumov) Syd. in Gäum) on the seed coat. Transfer of surface-disinfested seeds to potato dextrose agar and subsequent production of sclerotia showed that 2, 44, and 6% of the seed from each respective class were infested with S. sclerotiorum. One hundred seeds from each of these classes were planted into sterilized and nonsterilized soil at a rate of 5 seeds per pot. Toothpicks were placed to identify the location of each seed, and seeds were covered with 2 cm of soil. Pots were placed in growth chambers with a 14-h photoperiod under two temperature regimes: (i) at 20°C; and (ii) at 10°C for 10 days and then raised to 20°C. Soil was kept saturated by periodically top watering the pots for the first 10 days and bottom watering after that. Two weeks after planting, seeds were examined for formation of sclerotia and the percentages of seeds from which sclerotia were formed were calculated. The experiments were conducted four times. One to two (occasionally three) sclerotia were found in place of each seed that did not germinate. Sclerotia were mainly found from seeds of poor quality, with an average of 12% seeds that produced sclerotia. The frequency of sclerotia found in normal quality seeds was 0.4%, and no sclerotia were found from seeds with mycelial mats. The sclerotia were 2.36 ± 1.07 mm in width, 3.33 ± 1.11 mm in length, and 6.8 ± 3.7 mg in weight, with an averaged germination rate of 88% 8 months after production. Sclerotia production frequencies were 11.4 and 15.4% for temperature regimes (i) and (ii), respectively. Higher percentages of sclerotium production were found in sterilized soil (15.6%) than nonsterilized soil (7.5%). Our results indicate the possibility of internally infected soybean seeds as a means for field-to-field dissemination of S. sclerotiorum. References: (1) J. C. Tu. J. Phytopathol. 121:40, 1988. (2) X. B. Yang. ICM Newsl. 18, 1997.

Life-history strategy in Typhula incarnata and T. ishikariensis biotypes A, B, and C as determined by sclerotium production

The life-history strategies of Typhula incarnata and T. ishikariensis, the snow mold fungi, were studied in culture experiments. Radial mycelial growth at 0 °C, a temperature at which they thrive under natural conditions, was about half that of growth at the near-optimal temperature, 10 °C. However, when the colonies were covered with unsterile soil, mycelial growth at 0 °C was much greater than at 10 °C. Sclerotium production by T. incarnata was rapid in the latter half of the culture period, and sclerotium maturation was promoted by selected fungi. Typhula incarnata produced sclerotia frequently along the junction with colonies of selected fungi. Typhula ishikariensis biotype A produced the highest percent sclerotial biomass. Sclerotium maturation, i.e., pigmentation, was not stimulated by the selected fungi, but sclerotium production was moderately frequent in dual cultures with them. Typhula ishikariensis biotypes B and C produced the lowest percent sclerotial biomass. Their sclerotium maturation was rapid but not stimulated by the selected fungi. These findings suggest that although these snow mold fungi are, as a whole, stress tolerant, T. incarnata is relatively ruderal, T. ishikariensis biotype A is typically stress tolerant, and T. ishikariensis biotypes B and C are adapted to habitats with high stress and high disturbance.