First Report of Shoot Blight on Cryptomeria japonica Caused by Pestalotiopsis neglecta in China

Abstract

Cryptomeria japonica D. Don is widely distributed in Japan and China (Hosoo 2007). However, during a general survey conducted in 2012, many C. japonica trees showing symptoms of shoot blight were observed in Jiangsu and Yunnan provinces of China. Field observations were conducted from January 2013 to October 2014 in the two provinces. All of the 240 surveyed trees showing shoot blight were observed in the investigated areas. Diseased trees were characterized by the presence of green needles fading to yellow, with brown spots then developing on the needles, resulting in a brown to reddish-brown discoloration and wilt at the base or tip portion of the needles. A black halo developed at the junction between the diseased and healthy tissues. The needles withered and then the blight spread to the shoots, which finally died. There were abundant black acervuli present on the surface of the blight needles and shoots. Symptomatic tissues were collected from each site for isolation of the causative organism. Symptomatic tissues (0.5 cm) were surface disinfected in 0.1% HgCl₂ for 3 min and rinsed in distilled water three times, and three to five pieces of disinfected tissue were transferred onto a plate of potato dextrose agar (PDA) acidified with 0.2% (v/v) lactic acid; the plates were incubated in the dark at 25°C for 5 days. A species of fungus was consistently isolated from the symptomatic plants at all investigated sites. The colonies on PDA appeared with abundant white, aerial mycelia at first and showed wheel striations, but later the colonies turned pale yellow. The acervulus conidiomata (approximately 1 to 3 mm long) were covered by the mycelia at first and then broke out to reveal black scattered bodies with water droplets on the surface. The conidia were characterized as consisting of five cells, with a single hyaline basal appendage and two to five hyaline apical appendages. The conidia were fusiform, erect or slightly bent, thick-walled, 5.0 to 7.5 μm wide, and 20.0 to 27.0 μm long. The three median conidial cells were light to dark brown in color. Therefore, it was identified as Pestalotiopsis sp., based on the colony and conidial characteristics and morphology (Ge et al. 2009). Subsequently, it was used for inoculation of 8-year-old potted C. japonica trees. Ten wounded (by pricking with a sterile needle) and 10 unwounded shoots were inoculated (by spraying onto the canopy) separately with 10 ml of a conidial suspension (10⁶ conidia/ml). Ten wounded and 10 unwounded shoots of control trees were inoculated with sterile distilled water. Each group had three replicates. Each inoculated shoot was then covered with a piece of sterile plastic film for 48 h. All the inoculated trees were then transferred to and kept in a greenhouse at 25°C and 90% relative humidity. Observations were made every 2 days to score any symptoms of infection. Seven days after inoculation with Pestalotiopsis sp., 83.3% of the wounded and 76.7% of the nonwounded shoots appeared to show shoot blight symptoms similar to those of the naturally infected C. japonica trees in the field. None of the shoots in the control groups exhibited any symptoms. Pestalotiopsis sp. was reisolated from all the infected tissues of the inoculated shoots and identified as previously described, thus fulfilling Koch’s postulates. For further identification of the Pestalotiopsis sp., the internal transcribed spacer region was amplified with the primers ITS1/ITS4 as described by White et al. (1990). The polymerase chain reaction product was submitted to GenBank under the accession number MK305944. The phylogenetic relationship showed that the new sequence clustered with Pestalotiopsis neglecta, forming a separate clade distinct from other species in the Pestalotiopsis genus. To the best of our knowledge, this is the first report that P. neglecta causes shoot blight on C. japonica.