S. Matic

Comparative transcriptome profiling of resistant and susceptible rice genotypes in response to the seedborne pathogen Fusarium fujikuroi

BackgroundFusarium fujikuroi is the causal agent of bakanae, the most significant seed-borne disease of rice. Molecular mechanisms regulating defence responses of rice towards this fungus are not yet fully known. To identify transcriptional mechanisms underpinning rice resistance, a RNA-seq comparative transcriptome profiling was conducted on infected seedlings of selected rice genotypes at one and three weeks post germination (wpg).ResultsTwelve rice genotypes were screened against bakanae disease leading to the identification of Selenio and Dorella as the most resistant and susceptible cultivars, respectively. Transcriptional changes were more appreciable at 3 wpg, suggesting that this infection stage is essential to study the resistance mechanisms: 3,119 DEGs were found in Selenio and 5,095 in Dorella. PR1, germin-like proteins, glycoside hydrolases, MAP kinases, and WRKY transcriptional factors were up-regulated in the resistant genotype upon infection with F. fujikuroi. Up-regulation of chitinases and down-regulation of MAP kinases and WRKY transcriptional factors were observed in the susceptible genotype. Gene ontology (GO) enrichment analyses detected in Selenio GO terms specific to response to F. fujikuroi: ‘response to chitin’, ‘jasmonic acid biosynthetic process’, and ‘plant-type hypersensitive response’, while Dorella activated different mechanisms, such as ‘response to salicylic acid stimulus’ and ‘gibberellin metabolic process’, which was in agreement with the production of gibberellin A3 in Dorella plants.ConclusionsRNA-seq profiling was performed for the first time to analyse response of rice to F. fujikuroi infection. Our findings allowed the identification of genes activated in one- and three- week-old rice seedlings of two genotypes infected with F. fujikuroi. Furthermore, we found the pathways involved in bakanae resistance, such as response to chitin, JA-dependent signalling and hypersensitive response. Collectively, this provides important information to elucidate the molecular and cellular processes occurring in rice during F. fujikuroi infection and to develop bakanae resistant rice germplasm.

Biocontrol activity of an alkaline serine protease from Aureobasidium pullulans expressed in Pichia pastoris against four postharvest pathogens on apple

The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline serine protease could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short storage period.

Light affects fumonisin production in strains of Fusarium fujikuroi, Fusarium proliferatum, and Fusarium verticillioides isolated from rice

Three Fusarium species associated with bakanae disease of rice (Fusarium fujikuroi, Fusarium proliferatum, and Fusarium verticillioides) were investigated for their ability to produce fumonisins (FB1 and FB2) under different light conditions, and for pathogenicity. Compared to darkness, the conditions that highly stimulated fumonisin production were yellow and green light in F. verticillioides strains; white and blue light, and light/dark alternation in F. fujikuroi and F. proliferatum strains. In general, all light conditions positively influenced fumonisin production with respect to the dark. Expression of the FUM1 gene, which is necessary for the initiation of fumonisin production, was in accordance with the fumonisin biosynthetic profile. High and low fumonisin-producing F. fujikuroi strains showed typical symptoms of bakanae disease, abundant fumonisin-producing F. verticillioides strains exhibited chlorosis and stunting of rice plants, while fumonisin-producing F. proliferatum strains were asymptomatic on rice. We report that F. fujikuroi might be an abundant fumonisin producer with levels comparable to that of F. verticillioides and F. proliferatum, highlighting the need of deeper mycotoxicological analyses on rice isolates of F. fujikuroi. Our results showed for the first time the influence of light on fumonisin production in isolates of F. fujikuroi, F. proliferatum, and F. verticillioides from rice.

Identification of bakanae disease resistance loci in japonica rice through genome wide association study

BackgroundBakanae disease, caused by seed-borne Fusarium species, mainly F. fujikuroi, is a rice disease whose importance is considerably increasing in several rice growing countries, leading to incremental production losses.ResultsA germplasm collection of japonica rice was screened for F. fujikuroi resistance, allowing the identification of accessions with high-to-moderate levels of resistance to bakanae. A GWAS approach uncovered two genomic regions highly associated with the observed phenotypic variation for response to bakanae infection on the short arm of chromosome 1 (named as qBK1_628091) and on the long arm of chromosome 4 (named as qBK4_31750955). High levels of phenotypic resistance to bakanae were associated to the cumulated presence of the resistant alleles at the two resistance loci, suggesting that they can provide useful levels of disease protection in resistance breeding. A fine comparison with the genomic positions of qBK1_628091 and qBK4_31750955 with respect to the QTLs for bakanae resistance reported in the literature suggests that the resistant loci here described represent new genomic regions associated to F. fujikuroi resistance. A search for candidate genes with a putative role in bakanae resistance was conducted considering all the annotated genes and F. fujikuroi-related DEGs included in the two genomic regions highlighting several gene functions that could be involved in resistance, thus paving the way to the functional characterization of the resistance loci.ConclusionsNew effective sources for bakanae resistance were identified on rice chromosomes 1 and 4 and tools for resistance breeding are provided.

First Report of Damping off Caused by Pythium aphanidermatum on Bean (Phaseolus vulgaris) in Italy

During the summer 2014, symptoms of crown and root rot were observed on bean (Phaseolus vulgaris L.) cultivar Billo grown in a commercial field near Cuneo (northern Italy). Forty-day-old plants were stunted with leaf chlorosis and developed symptoms of root rot and necrotic streaks on the crown area. About 20 to 25% of plants out of 30,000 suddenly collapsed at temperatures ranging from 22 to 28°C. Fifty tissue fragments were excised from roots and basal stems of 20 plants, dipped in a solution containing 1% sodium hypochlorite, rinsed in sterile water, and plated on potato dextrose agar and on a semiselective medium for oomycetes (Masago et al. 1977). Plates were incubated under constant fluorescent light at 22 ± 1°C for 3 days. Ten out of the 40 colonies with abundant aerial mycelium obtained from both media were recovered and then plated on V8 medium. Under light microscope, aseptate hyphae, 3.57 to 7.8 µm (mean 5.9 µm) wide were observed. Oogonia were globose, smooth, and measured 12.6 to 28.0 µm (mean 22.1 µm). Antheridia were barrel-shaped (8.2 to10.3 µm), and oospores were 14.8 to 22.1 µm (mean 19.3 µm) in diameter. These morphological characters identified the microorganism as a Pythium sp. (Spencer 2005). The internal transcribed spacer (ITS) region of rDNA of a single isolate (Py 13/14) was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis of the 777-bp segment showed a 100% similarity with the sequence of Pythium aphanidermatum (GenBank accession KY095191). The nucleotide sequence was deposited in GenBank under accession number MF040822. Pathogenicity tests were performed twice on bean cultivar Billo. Pots, containing 2 liters of steam-disinfested organic peat substrate, were infested with wheat and hemp kernels colonized with Py 13/14 strain of P. aphanidermatum at a rate of 2 g/liter. Five seeds/pot were sown 48 h after inoculation in six pots filled with the infested medium, and the same number of seeds was sown in uninfested substrate. Plants were kept in a greenhouse at 22 to 27°C under 12 h of photoperiod. Preemergence damping-off was observed in 37 to 43% of plants 10 days after the artificial inoculation. After 20 days, 47 to 60% of plants were infected, showing the same symptoms as previously described. Control plants remained healthy. P. aphanidermatum was consistently reisolated with 80% frequency from the lesions of the root and crown of the plants. To our knowledge, this is the first report of the presence of P. aphanidermatum on bean in Italy. The same pathogen has also been reported on bean in Spain and in Oman (Al-Mahmooli et al. 2015; Serrano et al. 2008). Due to the economic importance of beans in Piedmont (Italy) in the Cuneo province, where about 5,000 ha are grown in open field generally in monoculture, the spread of the disease could cause serious damage in this area as well as elsewhere.

FIRST REPORT OF DOWNY MILDEW (PLASMOPARA OBDUCENS) ON IMPATIENS WALLERIANA IN ITALY

During summer 2012, potted plants of impatiens (Impatiens walleriana) grown in gardens near Biella (northern Italy) showed symptoms of downy mildew. Infected leaves were paler green than normal and showed white, downy growth on the lower surface. Plants collapsed very rapidly, especially at high relative humidity (RH). Microscopical observations of infected leaves, maintained for 24 h at high RH, disclosed the presence of hyaline, tree-like, straight, 120-350×5.8-10 μm sporangiophores, with three sterigma. Sporangia were ovoid, hyaline and 10.7-15.4×11.7-16.6 (average 13.2×14.5) μm in size. Oospores were not observed in leaf tissue. The DNA region encoding for the large ribosomal subunit (LSU rDNA) was amplified using primers NL1 and NL4 (Maier et al., 2003) and sequenced (GenBank accession No. JX880252LSU). BLAST analysis of the 729 bp product obtained showed a similarity of 99% (E-value=0) with Plasmopara obducens from the USA (GenBank accession No. JX217746). To confirm pathogenicity, 60-day-old impatiens plants, grown singly in 15 litre pots in a growth chamber at 20±1°C, were inoculated by spraying leaves with a suspension of 1×105 sporangia/ml. Control plants were sprayed with distilled water. Plants were covered with plastic bags for 4 days. The first symptoms (chlorosis) developed 8 days post inoculation. Control plants remained healthy. This is the first report of P. obducens in Italy. The disease has been reported in several countries, including the USA (Wegulo et al., 2004), UK (Lane et al., 2005) and Serbia (Bulajic et al., 2011). Currently, this disease is present in several gardens in northern Italy, where its importance may increase rapidly due to the widespread cultivation of impatiens.

Combined Effect of CO 2 and Temperature on Wheat Powdery Mildew Development

The effect of simulated climate changes by applying different temperatures and CO2 levels was investigated in the Blumeria graminis f. sp. tritici/wheat pathosystem. Healthy and inoculated plants were exposed in single phytotrons to six CO2+temperature combinations: (1) 450 ppm CO2/18–22°C (ambient CO2 and low temperature), (2) 850 ppm CO2/18–22°C (elevated CO2 and low temperature), (3) 450 ppm CO2/22–26°C (ambient CO2 and medium temperature), (4) 850 ppm CO2/22–26°C (elevated CO2 and medium temperature), (5) 450 ppm CO2/26–30°C (ambient CO2 and high temperature), and (6) 850 ppm CO2/26–30°C (elevated CO2 and high temperature). Powdery mildew disease index, fungal DNA quantity, plant death incidence, plant expression of pathogenesis-related (PR) genes, plant growth parameters, carbohydrate and chlorophyll content were evaluated. Both CO2 and temperature, and their interaction significantly influenced powdery mildew development. The most advantageous conditions for the progress of powdery mildew on wheat were low temperature and ambient CO2. High temperatures inhibited pathogen growth independent of CO2 conditions, and no typical powdery mildew symptoms were observed. Elevated CO2 did not stimulate powdery mildew development, but was detrimental for plant vitality. Similar abundance of three PR transcripts was found, and the level of their expression was different between six phytotron conditions. Real time PCR quantification of Bgt was in line with the disease index results, but this technique succeeded to detect the pathogen also in asymptomatic plants. Overall, future global warming scenarios may limit the development of powdery mildew on wheat in Mediterranean area, unless the pathogen will adapt to higher temperatures.

First report of leaf smut caused by Entyloma gaillardianum on Gaillardia aristata in Italy.

Blanketflower (Gaillardia aristata) is a popular flowering plant commonly used in parks and gardens, belonging to Asteraceae. Starting in May 2017, 6-month-old plants grown in a private garden located near Biella (latitude, 45°36′00″N; longitude, 8°03′00″E) in Northern Italy showed signs and severe symptoms of a previously unknown disease. About 70% of the 30 G. aristata plants were affected, exhibiting circular spots on leaves, from 0.8 to 1.3 cm in diameter, sometimes coalescent. Amphigenous spots were light green and 6 to 9 days later turned brown and necrotic. Microscopic examination of leaf tissue sections showed sori composed by massive double-walled hyaline to yellowish spores in the intercellular spaces of the host tissue, ranging from 9.8 to 12.3 μm (mean, 10.5 μm) in diameter, typical of the teliospores of the genus Entyloma (Vanky 1982). Hyaline, cylindrical, nonseptate conidia from straight to curved, 12.5 to 19.5 × 2.5 to 3.8 μm, of the anamorphic stage were also observed. DNA was extracted from sori by using the E.Z.N.A. Fungal DNA Mini Kit (Omega BioTek). The internal transcribed spacer (ITS) regions of rDNA were amplified using the primers ITS1/ITS4 (White et al. 1990) and sequenced at the BMR Genomics Centre (Padua, Italy). The 442-bp product of this isolate (IT62) was deposited at GenBank (accession no. MF521597), and a BLASTn search showed a 100% similarity with Entyloma gaillardianum (accession no. AY081037). Pathogenicity was confirmed by spraying a spore suspension (5 × 10⁴/ml), prepared from homogenized infested leaves, on three potted (3 liter) plants of G. aristata, and the same number of control plants were sprayed with sterile water. Plants were kept in a growth chamber at 21 to 24°C under 100% relative humidity for 5 days. All inoculated plants developed typical spots after 10 days, showing sori and spores similar to those previously described, whereas no symptoms developed on the control plants. Pathogenicity tests were carried out twice with the same results. Leaf smut caused by E. gaillardianum was reported on G. aristata in Germany, Israel, Romania, and Ukraine (Farr and Rossman 2017) and on Gaillardia × grandiflora in North America (Glawe et al. 2010). The presence of this pathogen is critical because Entyloma sp. causes severe economic losses on many different Asteraceae hosts, including Gaillardia sp. This is, to our knowledge, the first report of E. gaillardianum on G. aristata in Italy.

Molecular characterization and sensitivity to demethylation inhibitor fungicides of Aspergillus fumigatus from orange-based compost

Aspergillus fumigatus, the causal agent of human aspergilloses, is known to be non-pathogenic in plants. It is present as saprophyte in different types of organic matter and develops rapidly during the high-temperature phase of the composting process. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides and resistant isolates have been recently reported. The present study aims to estimate the abundance, genetic diversity and DMI sensitivity of A. fumigatus during the composting process of orange fruits. Composting of orange fruits resulted in a 100-fold increase in A. fumigatus frequency already after 1 week, demonstrating that the degradation of orange fruits favoured the growth of A. fumigatus in compost. Most of A. fumigatus isolates belonged to mating type 2, including those initially isolated from the orange peel, whereas mating type 1 evolved towards the end of the composting process. None of the A. fumigatus isolates expressed simultaneously both mating types. The 52 investigated isolates exhibited moderate SSR polymorphisms by formation of one major (47 isolates) and one minor cluster (5 isolates). The latter included mating type 1 isolates from the last sampling and the DMI-resistant reference strains. Only few isolates showed cyp51A polymorphisms but were sensitive to DMIs as all the other isolates. None of the A. fumigatus isolates owned any of the mutations associated with DMI resistance. This study documents a high reproduction rate of A. fumigatus during the composting process of orange fruits, requesting specific safety precautions in compost handling. Furthermore, azole residue concentrations in orange-based compost were not sufficient to select A. fumigatus resistant genotypes.

Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification of Fusarium fujikuroi in Rice Plants and Seeds

Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.