Detection and quantification of black foot and crown and root rot pathogens in grapevine nursery soils in the Western Cape of South Africa

Black foot disease (BFD) and crown and root rot (CRR) are important soilborne diseases that affect young grapevines in nurseries and vineyards. A 3-year survey (2013–2015) of five open-field grapevine nurseries was conducted in the Western Cape Province of South Africa. The survey involved the isolation of BFD and CRR pathogens from grafted rootstocks (ten plants per nursery, per year) that were rooted in soil for 1 year. In 2013 and 2015, grapevines were sampled, while in 2014, sampling was focused on rotation crops and weeds (ten plants each). The rotation crops included white mustard, lupins, canola, triticale and forage radish. The weed species sampled included Johnson grass, ryegrass, winter grass, Cape marigold and corn spurry. Soil samples from ten sites per nursery were also collected in close proximity to the sampled plants, at depths of 0–30 cm and 30–60 cm (ten samples per depth). Isolations were made from the grapevines, rotation crops and weeds. Pathogen detection and quantification in the soil were determined using quantitative real-time polymerase chain reaction technology. The predominant BFD pathogens isolated from grapevines were Campylocarpon fasciculare, Ca. pseudofasciculare and Dactylonectria macrodidyma. The predominant CRR pathogens were Pythium irregulare and Phytopythium vexans. Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, Py. irregulare, Py. ultimum var. ultimum and Py. heterothallicum were isolated from triticale roots. Dactylonectria spp. were also isolated from corn spurry, while Py. irregulare and Py. ultimum var. ultimum were isolated from numerous weeds and rotation crops. Mean soil DNA concentrations of Ilyonectria and Dactylonectria were from 0.04 to 37.14 pg μL-1, and for Py. irregulare were between 0.01 and 3.77 pg μL-1. The Phytophthora mean soil DNA concentrations ranged from 0.01 to 33.48 pg μL-1. The qPCR protocols successfully detected and quantified BFD and CRR pathogens in grapevine nursery soil. This is the first report of D. pauciseptata and D. alcacerensis in South African grapevine nurseries

Detection and quantification of black foot and crown and root rot pathogens in grapevine nursery soils in the Western Cape of South Africa

CITATION: Langenhoven, S., et al. 2018. Detection and quantification of black foot and crown and root rot pathogens in grapevine nursery soils in the Western Cape of South Africa. Phytopathologia Mediterranea, 57(3):519-537, doi:10.14601/Phytopathol_Mediterr-23921.The original publication is available at http://www.fupress.netBlack foot disease (BFD) and crown and root rot (CRR) are important soilborne diseases that affect young grapevines in nurseries and vineyards. A 3-year survey (2013–2015) of five open-field grapevine nurseries was conducted in the Western Cape Province of South Africa. The survey involved the isolation of BFD and CRR pathogens from grafted rootstocks (ten plants per nursery, per year) that were rooted in soil for 1 year. In 2013 and 2015, grapevines were sampled, while in 2014, sampling was focused on rotation crops and weeds (ten plants each). The rotation crops included white mustard, lupins, canola, triticale and forage radish. The weed species sampled included Johnson grass, ryegrass, winter grass, Cape marigold and corn spurry. Soil samples from ten sites per nursery were also collected in close proximity to the sampled plants, at depths of 0–30 cm and 30–60 cm (ten samples per depth). Isolations were made from the grapevines, rotation crops and weeds. Pathogen detection and quantification in the soil were determined using quantitative real-time polymerase chain reaction technology. The predominant BFD pathogens isolated from grapevines were Campylocarpon fasciculare, Ca. pseudofasciculare and Dactylonectria macrodidyma. The predominant CRR pathogens were Pythium irregulare and Phytopythium vexans. Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, Py. irregulare, Py. ultimum var. ultimum and Py. heterothallicum were isolated from triticale roots. Dactylonectria spp. were also isolated from corn spurry, while Py. irregulare and Py. ultimum var. ultimum were isolated from numerous weeds and rotation crops. Mean soil DNA concentrations of Ilyonectria and Dactylonectria were from 0.04 to 37.14 pg μL-1, and for Py. irregulare were between 0.01 and 3.77 pg μL-1. The Phytophthora mean soil DNA concentrations ranged from 0.01 to 33.48 pg μL-1. The qPCR protocols successfully detected and quantified BFD and CRR pathogens in grapevine nursery soil. This is the first report of D. pauciseptata and D. alcacerensis in South African grapevine nurseries.http://www.fupress.net/index.php/pm/article/view/23921Publisher's versio

Characterization of the Mycovirome of the Phytopathogenic Fungus, Neofusicoccum parvum

Neofusicoccum parvum is a fungal plant-pathogen belonging to the family Botryosphaeriaceae, and is considered one of the most aggressive causal agents of the grapevine trunk disease (GTD) Botryosphaeria dieback. In this study, the mycovirome of a single strain of N. parvum (COLB) was characterized by high throughput sequencing analysis of total RNA and subsequent bioinformatic analyses. Contig annotations, genome completions, and phylogenetic analyses allowed us to describe six novel mycoviruses belonging to four different viral families. The virome is composed of two victoriviruses in the family Totiviridae, one alphaendornavirus in the family Endornaviridae, two mitoviruses in the family Mitoviridae, and one narnavirus belonging to the family Narnaviridae. The presence of the co-infecting viruses was confirmed by sequencing the RT-PCR products generated from total nucleic acids extracted from COLB. This study shows that the mycovirome of a single N. parvum strain is highly diverse and distinct from that previously described in N. parvum strains isolated from grapevines

Comparative Study of Secreted Proteins, Enzymatic Activities of Wood Degradation and Stilbene Metabolization in Grapevine Botryosphaeria Dieback Fungi

Botryosphaeriaceae fungi are plant pathogens associated with Botryosphaeria dieback. To better understand the virulence factors of these fungi, we investigated the diversity of secreted proteins and extracellular enzyme activities involved in wood degradation and stilbene metabolization in Neofusicoccum parvum and Diplodia seriata, which are two major fungi associated with grapevine B. dieback. Regarding the analysis of proteins secreted by the two fungi, our study revealed that N. parvum, known to be more aggressive than D. seriata, was characterized by a higher quantity and diversity of secreted proteins, especially hydrolases and oxidoreductases that are likely involved in cell wall and lignin degradation. In addition, when fungi were grown with wood powder, the extracellular laccase and Mn peroxidase enzyme activities were significantly higher in D. seriata compared to N. parvum. Importantly, our work also showed that secreted Botryosphaeriaceae proteins produced after grapevine wood addition are able to rapidly metabolize the grapevine stilbenes. Overall, a higher diversity of resveratrol and piceatannol metabolization products was found with enzymes of N. parvum compared to D. seriata. This study emphasizes the diversity of secreted virulence factors found in B. dieback fungi and suggests that some resveratrol oligomers produced in grapevine wood after pathogen attack could be formed via pathogenic fungal oxidases. Keywords: Botryosphaeriaceae; grapevine; stilbene metabolization; secreted protein

Grapevine Botryosphaeria dieback fungi have specific aggressiveness factor repertory involved in wood decay and stilbene metabolization

<div><p>Grapevine trunk diseases: Eutypa dieback, esca and Botryosphaeria dieback, which incidence has increased recently, are associated with several symptoms finally leading to the plant death. In the absence of efficient treatments, these diseases are a major problem for the viticulture; however, the factors involved in disease progression are not still fully identified. In order to get a better understanding of Botryosphaeria dieback development in grapevine, we have investigated different factors involved in <i>Botryosphaeriaceae</i> fungi aggressiveness. We first evaluated the activity of the wood-degrading enzymes of different isolates of <i>Neofusicoccum parvum</i> and <i>Diplodia seriata</i>, two major fungi associated with Botryosphaeria dieback. We further examinated the ability of these fungi to metabolize major grapevine phytoalexins: resveratrol and δ-viniferin. Our results demonstrate that <i>Botryosphaeriaceae</i> were characterized by differential wood decay enzymatic activities and have the capacity to rapidly degrade stilbenes. <i>N</i>. <i>parvum</i> is able to degrade parietal polysaccharides, whereas <i>D</i>. <i>seriata</i> has a better capacity to degrade lignin. Growth of both fungi exhibited a low sensitivity to resveratrol, whereas δ-viniferin has a fungistatic effect, especially on <i>N</i>. <i>parvum</i> Bourgogne S-116. We further show that <i>Botryosphaeriaceae</i> are able to metabolize rapidly resveratrol and δ-viniferin. The best stilbene metabolizing activity was measured for <i>D</i>. <i>seriata</i>. In conclusion, the different <i>Botryosphaeriaceae</i> isolates are characterized by a specific aggressiveness repertory. Wood and phenolic compound decay enzymatic activities could enable <i>Botryosphaeriaceae</i> to bypass chemical and physical barriers of the grapevine plant. The specific signature of <i>Botryosphaeriaceae</i> aggressiveness factors could explain the importance of fungi complexes in synergistic activity in order to fully colonize the host.</p></div

Resveratrol metabolization by <i>Botryosphaeriaceae</i> fungi.

<p>Resveratrol (initially added at 50 μM) was quantified by LC-MS in PDA medium extract of <i>N</i>. <i>parvum</i> Bourgogne S-116, <i>N</i>. <i>parvum</i> Bt-67 and <i>D</i>. <i>seriata</i> 98.1. The resveratrol quantity is expressed in relative quantity (% of control) compared to control medium without fungi at different time points (3, 6 and 11 days; A), or when fungi have saturated 95% of the Petri Dish surface (B). Values are means and SD of three biological replicates, each calculated from the mean of three technical replicates. Means with a same letter are not significantly different at <i>p</i>≤0.05 (Tukey Contrasts).</p

Effect of resveratrol on <i>Botryosphaeriaceae</i>.

<p>The resveratrol was tested at a final concentration of 50 μM (grey lines) and 250 μM (gray dotted lines) on mycelial growth of <i>N</i>. <i>parvum</i> Bourgogne S-116 (A), <i>N</i>. <i>parvum</i> Bt-67 (B) and <i>D</i>. <i>seriata</i> 98.1 (C). Negative control was performed by adding DMSO instead of resveratrol. Values are means and SD of two biological replicates, each calculated from the mean of three technical replicates. Means with a * are significantly different from control at <i>p</i> ≤ 0,05 (Tukey Contrasts).</p

Effect of δ-viniferin on <i>Botryosphaeriaceae</i>.

<p>The δ-viniferin was tested at a final concentration of 50 μM (grey lines) and 250 μM (gray dotted lines) on <i>N</i>. <i>parvum</i> Bourgogne S-116 (A), <i>N</i>. <i>parvum</i> Bt-67 (B) and <i>D</i>. <i>seriata</i> 98.1 (C) growth. δ-viniferin was dissolved in DMSO and negative control was performed by adding DMSO alone. Values are means and SD of two biological replicates, each calculated from the mean of three technical replicates. Means with a * are significantly different from control at <i>p</i> ≤ 0.05 (Tukey Contrasts).</p

Polysaccharide degrading enzymatic activities.

<p>Glucose liberation resulting from cellulase, hemicellulase and total activities of secreted proteins from <i>N</i>. <i>parvum</i> Bourgogne S-116 (A), <i>N</i>. <i>parvum</i> Bt-67 (B) and <i>D</i>. <i>seriata</i> 98.1 (C), was measured with a colorimetric method using dinitrosalicylic acid (DNS). Total proteins were isolated from culture filtrates of fungi grown in liquid malt medium (Malt), malt medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (Malt + W), Erickson and Peterson medium (EP) and Erickson and Peterson medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (EP + W). Values are means and SD of three biological replicates, each calculated from the mean of three technical replicates. Means with a same letter are not significantly different at <i>p</i>≤0.05 (Tukey Contrasts).</p

Grapevine Botryosphaeria dieback fungi have specific aggressiveness factor repertory involved in wood decay and stilbene metabolization - Fig 2

<p><b>(A) Laccase activity.</b> Oxidation of ABTS in the radical cation ABTS^.+ was measured with total secreted proteins from <i>N</i>. <i>parvum</i> Bourgogne S-116, <i>N</i>. <i>parvum</i> Bt-67 and <i>D</i>. <i>seriata</i> 98.1. Total proteins were isolated from culture filtrates of fungi grown in malt medium (Malt), malt medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (Malt + W), Erickson and Peterson medium (EP) and Erickson and Peterson medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (EP + W). <b>(B) Manganese peroxidase activity.</b> Oxidation coupling of MBTH/DMAB was measured in presence of H₂O₂ and Mn²⁺. Total proteins were isolated from culture filtrates of fungi grown in liquid malt medium (Malt), malt medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (Malt + W), Erickson and Peterson medium (EP) and Erickson and Peterson medium supplemented with <i>V</i>. <i>vinifera</i> sawdust (EP + W). Values are means and SD of three biological replicates, each calculated from the mean of two technical replicates. Means with a same letter are not significantly different at <i>p</i>≤0,05 (Tukey Contrasts).</p