Quantification of Phytophthora pisi DNA and RNA transcripts during in planta infection of pea

Abstract

Phytophthora pisi is a novel species of Phytophthora that causes root rot in pea and is a potentially devastating pathogen for the cultivation of pea in many parts of the world. The focus of the current study was to: (a) develop an in planta infection system of pea seedling roots; (b) develop a quantitative PCR method to measure P. pisi and pea DNA; and (c) test for differential gene expression of selected putative pathogenicity factors in P. pisi during infection of pea. P. pisi zoospores were attracted to the tips of the pea seedling roots and encysted within 30 min. After 6 h the pathogen had reached about five cortical cell layers using both interand intracellular growth, and water-soaked lesions were observed visually after 20 h. DNA-based detection of P. pisi and pea showed a gradual accumulation of P. pisi DNA in pea root tips up to 48 h post infection. Quantitative reverse transcriptase PCR showed induction of genes encoding putative enzyme inhibitors during the early phase of infection, between 2 h and 6 h post infection. Genes encoding putative cysteine protease, glucanase, pleiotropic drug transporter and crinkler proteins were induced during the late phase of infection. The induced P. pisi cysteine protease belonged to a cathepsin-L-like group from the C1A subfamily of peptidases and displayed a unique modular structure that included a MD-2-related lipid-recognition domain.

A Rapid and Efficient Method for In Vitro Screening of Taro for Leaf Blight Disease Caused by Phytophthora colocasiae

“…Lima bean broth in combination with autoclaved river water was used to produce Phytophthora pisi zoospores (1.2 × 10 5 zoospores/ml) (Hosseini et al. ). However, sporulation and zoospore release occur synchronously in diseased plants, but asynchronously in laboratory cultures (Judelson and Blanco ).…”

“…Ahonsi et al (2007) developed an in vitro 'wet plate' method for producing Phytophthora nicotianae zoospores (1 9 10 6 zoospores/ml) after testing different methods using clarified V8 media/broth that resulted in consistently low yields of zoospores, while Kong et al (2003) produced P. nicotianae zoospores (1 9 10 4 zoospores/ml) by incubating finely chopped mycelium plugs bearing numerous sporangia for 2 to 4 h in 25 ml of prechilled sterile soil water extract (SSWE) under fluorescent light. Lima bean broth in combination with autoclaved river water was used to produce Phytophthora pisi zoospores (1.2 9 10 5 zoospores/ml) (Hosseini et al 2012). However, Fig.…”

Zoospore chemotaxis of closely related legume‐root infecting Phytophthora species towards host isoflavones

“…The degree of chemotactic attraction of zoospores of different isolates of P. pisi , P. niederhauserii , P. vignae , one isolate of P. sojae (Table ) and A. euteiches (isolate 11k3) to each dilution of the three isoflavones was assayed. Zoospores were produced as described previously (Hosseini et al ., ) and filtered through Miracloth (Calbiochem). A 1 μL glass capillary tube (Drummond) was filled with the dilution of each iso‐flavone and sealed with vaseline at one end to keep the liquid in the tube.…”

“…The distal ends of the roots were cut 3 cm from the tips. Zoospores from one strain of each Phytophthora species (Table ) were produced as described previously (Hosseini et al ., ) and filtered through Miracloth (Calbiochem). Eight‐well plastic incubation trays (Nalge Nunc International) were used for the assay, in which one detached root was placed horizontally at the bottom of each well, followed by the addition of the zoospore suspension (1·3 × 10 5 zoospores mL −1 ).…”

An efficient method for zoospore production, infection and real-time quantification of Phytophthora cajani causing Phytophthora blight disease in pigeonpea under elevated atmospheric CO2

“…Various methods reported for screening Phytophthora in different crops included; in vitro leaf disk infection of brussels sprout plants ( Brassica oleracea var. gemmifera ) by infectious zoospores of P. brassicae [ 14 ]; root dip of pea in zoospore suspension of P. pisi [ 29 ]; leaf detached method in Nicotiana tabacum for P. parasitica var. nicotianae [ 11 ]; germination of soybean seeds in soil supplemented with zoospores suspension of P. megasperma var.…”