Involvement of miRNAs in Metabolic Herbicide Resistance to Bispyribac-Sodium in Echinochloa crus-galli (L.) P. Beauv.

Several mechanisms involved in weed herbicide resistance are unknown, particularly those acting at the epigenetic level, such as the capacity of small-non-coding RNAs (sncRNAs) to target messenger RNAs of genes involved in herbicide detoxification. The transcription of these sncRNAs is stimulated by epigenetic factors, thereby affecting gene expression. This study was carried out in order to evaluate, for the first time in Echinochloa crus-galli (L.) P. Beauv. (barnyardgrass), the capacity of miRNAs to regulate the expression of genes associated with bispyribac-sodium detoxification. The expression profiles of eight miRNAs with a high degree of complementarity (≥80%) with mRNAs of genes involved in herbicide detoxification (CYP450, GST and eIF4B) were determined by qRT-PCR before and after herbicide spraying. Five of the miRNAs studied (gra-miR7487c, gma-miR396f, gra-miR8759, osa-miR395f, ath-miR847) showed an increased expression after herbicide application in both susceptible and resistant biotypes. All the miRNAs, except gra-miR8759, were more highly expressed in the herbicide-resistant biotypes. In specimens with increased expression of miRNAs, we observed reduced expression of the target genes. The remaining three miRNAs (ata-miR166c-5p, ath-miR396b-5p and osa-miR5538) showed no over-expression after herbicide treatment, and no difference in expression was recorded between susceptible and resistant biotypes. Our results represent a first overview of the capacity of miRNAs to regulate the expression of genes involved in bispyribac-sodium detoxification in the genus Echinochloa. Further research is required to identify novel miRNAs and target genes to develop more focused and sustainable strategies of weed control

Incidence of resistance to ALS and ACCase inhibitors in Echinochloa species and soil microbial composition in Northern Italy

Abstract The increasing amount of weeds surviving herbicide represents a very serious problem for crop management. The interaction between microbial community of soil and herbicide resistance, along with the potential evolutive consequences, are still poorly known and need to be investigated to better understand the impact on agricultural management. In our study, we analyzed the microbial composition of soils in 32 farms, located in the Northern Italy rice-growing area (Lombardy) with the aim to evaluate the relationship between the microbial composition and the incidence of resistance to acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibiting herbicides in Echinochloa species. We observed that the coverage of weeds survived herbicide treatment was higher than 60% in paddy fields with a low microbial biodiversity and less than 5% in those with a high microbial biodiversity. Fungal communities showed a greater reduction in richness than Bacteria. In soils with a reduced microbial diversity, a significant increase of some bacterial and fungal orders (i.e. Lactobacillales, Malasseziales and Diaporthales) was observed. Interestingly, we identified two different microbial profiles linked to the two conditions: high incidence of herbicide resistance (H-HeR) and low incidence of herbicide resistance (L-HeR). Overall, the results we obtained allow us to make hypotheses on the greater or lesser probability of herbicide resistance occurrence based on the composition of the soil microbiome and especially on the degree of biodiversity of the microbial communities

An Improved Method for Assessing Simple Sequence Repeat (SSR) Variation in Echinochloa crus-galli (L.) P. Beauv (Barnyardgrass)

Echinochloa crus-galli (L.) P. Beauv. (barnyardgrass) is one of the most noxious weeds infesting Italian rice fields. It is characterized by high genetic intraspecific variability and has developed resistance to several classes of herbicides. The aim of our study was to assess, for the first time in Italy, the genetic diversity in E. crus-galli from differently managed rice fields in the Lombardy region (Northern Italy) using eight specific SSR markers. To this purpose, an amplification protocol was optimized, testing different DNA concentrations, PCR mixtures, and temperatures. A total of 48 alleles were identified in 144 samples. SSR fingerprint analysis using R 3.6.3 software (poppr, polysat, and StAMPP) allowed us to handle SSRs as codominant and polyploid data. The results suggested that genetic richness and diversity were high. The analysis of molecular variance (AMOVA) indicated that genetic variation exists mainly between agronomic managements (47.23%) and among populations (37.01%). Hierarchical clustering and PCoA were in concordance with the identification of four distinct genetic groups. Our results confirm that SSR markers represent a valuable and affordable tool for the assessment of E. crus-galli genetic diversity and would grant useful information to plan more targeted, effective, and sustainable control strategies against barnyardgrass. The improved methodology applied here allowed us to assess the genetic variability of an allo-hexaploid species without information loss and biased results

An Improved Method for Assessing Simple Sequence Repeat (SSR) Variation in <i>Echinochloa crus-galli</i> (L.) P. Beauv (Barnyardgrass)

Echinochloa crus-galli (L.) P. Beauv. (barnyardgrass) is one of the most noxious weeds infesting Italian rice fields. It is characterized by high genetic intraspecific variability and has developed resistance to several classes of herbicides. The aim of our study was to assess, for the first time in Italy, the genetic diversity in E. crus-galli from differently managed rice fields in the Lombardy region (Northern Italy) using eight specific SSR markers. To this purpose, an amplification protocol was optimized, testing different DNA concentrations, PCR mixtures, and temperatures. A total of 48 alleles were identified in 144 samples. SSR fingerprint analysis using R 3.6.3 software (poppr, polysat, and StAMPP) allowed us to handle SSRs as codominant and polyploid data. The results suggested that genetic richness and diversity were high. The analysis of molecular variance (AMOVA) indicated that genetic variation exists mainly between agronomic managements (47.23%) and among populations (37.01%). Hierarchical clustering and PCoA were in concordance with the identification of four distinct genetic groups. Our results confirm that SSR markers represent a valuable and affordable tool for the assessment of E. crus-galli genetic diversity and would grant useful information to plan more targeted, effective, and sustainable control strategies against barnyardgrass. The improved methodology applied here allowed us to assess the genetic variability of an allo-hexaploid species without information loss and biased results

Involvement of miRNAs in Metabolic Herbicide Resistance to Bispyribac-Sodium in <i>Echinochloa crus-galli</i> (L.) P. Beauv.

Several mechanisms involved in weed herbicide resistance are unknown, particularly those acting at the epigenetic level, such as the capacity of small-non-coding RNAs (sncRNAs) to target messenger RNAs of genes involved in herbicide detoxification. The transcription of these sncRNAs is stimulated by epigenetic factors, thereby affecting gene expression. This study was carried out in order to evaluate, for the first time in Echinochloa crus-galli (L.) P. Beauv. (barnyardgrass), the capacity of miRNAs to regulate the expression of genes associated with bispyribac-sodium detoxification. The expression profiles of eight miRNAs with a high degree of complementarity (≥80%) with mRNAs of genes involved in herbicide detoxification (CYP450, GST and eIF4B) were determined by qRT-PCR before and after herbicide spraying. Five of the miRNAs studied (gra-miR7487c, gma-miR396f, gra-miR8759, osa-miR395f, ath-miR847) showed an increased expression after herbicide application in both susceptible and resistant biotypes. All the miRNAs, except gra-miR8759, were more highly expressed in the herbicide-resistant biotypes. In specimens with increased expression of miRNAs, we observed reduced expression of the target genes. The remaining three miRNAs (ata-miR166c-5p, ath-miR396b-5p and osa-miR5538) showed no over-expression after herbicide treatment, and no difference in expression was recorded between susceptible and resistant biotypes. Our results represent a first overview of the capacity of miRNAs to regulate the expression of genes involved in bispyribac-sodium detoxification in the genus Echinochloa. Further research is required to identify novel miRNAs and target genes to develop more focused and sustainable strategies of weed control

Phylogenetic Comparison between Italian and Worldwide Hericium Species (Agaricomycetes)

ABSTRACT: A broad literature concerns the genus Hericium, mainly regarding the medicinal properties of H. erinaceus. Congeneric species of H. erinaceus have been poorly investigated. We collected basidiomata of H. alpestre, H. coralloides and H. erinaceus in Italy and isolated the corresponding mycelia in pure culture. Analysis of the respective internal transcribed spacer regions confirmed the morphological identification of the strains. Internal transcribed spacer sequences from the Italian strains were phylogenetically compared along with 64 other sequences available from GenBank, the CBS Strain Database, and the European Nucleotide Archive (ENA) for the same Hericium species. Geographic origin and host plant species were cross-checked using the above data banks. Bayesian phylogenetic analysis produced a phylogram that permitted good discrimination among Hericium species. It provides an updated phylogeny within the genus Hericium and a better understanding of affinity among the species analyzed. The main Hericium clade includes the following: the H. erinaceus group and the H. alpestre/H. coralloides group, where the two species cluster separately. This study also allowed us to differentiate the H. erinaceus group on a biogeographical basis. The phylogenetic comparison further confirms the importance of a joint morphological-molecular approach to avoid misidentification and to guarantee the quality of strains for further chemical and medicinal characterization