Introduction

Walnut (Juglans regia), is an indigenous tree species in China. In Shaanxi province in the northwest region of the country, market demand has stimulated the importation of new cultivars from adjacent provinces (Xinjiang, Gansu, Henan, Sichuan, Yunnan), as well as from the US and the EU. It was in this context that cultivar 'Xiangling' was brought into the northwest region from Xinjiang province. Seedlings of Juglans regia are typically grown to serve as rootstocks for scions of walnut cultivars. In 2010, disease symptoms, including lesions girdling the base of the stems, were observed on one-to three-year-old seedlings grafted with cv. "Xiangling" as scions. Cankers followed and these were accompanied by brown and red-brown exudates, Fig. 1a. Finally, the seedlings died.

The only known canker diseases affecting walnuts in China are Valsa and Botryosphaeria canker. The disease caused by Valsa juglandis is called locally known as "Black Water Disease" and often leads to plantation failure. Botryosphaeria canker caused by B. dothidea (anamorph: Fusicoccum aesculi) appears later on the same trees affected by Valsa. The species of Botryosphaeria associated with this walnut canker do vary around the world (Hagsag et al. 2007; Rumbos 1987; Yu et al. 2010). But in all cases, only adult trees are affected, and its pathogen is believed to be Botryosphaeria dothidea; while in Egypt the pathogen is Botryodiplodia theobromae (Hagsag et al. 2007) and in Greece, it is B. ribis (Rumbos 1987). Some authors consider B. ribis as a synonym of B. Dothidea (von Arx and Muller 1975; Maas and Uecker 1984), while others think not (Phillips et al. 2002; Yu et al. 2010). However, both are reported occurring only on adult trees.

Materials and methods Morphology and pathogenesis analysis

Tissue samples from 10 walnut seedling stems were cultured on medium of potato, dextrose and agar (abv. PDA), following surface-sterilization in 1/1000 g/L mercury bichloride for 30 s followed by washing in sterile water. The samples were then incubated at 25 ℃ in a dark room. Other samples from adult trees in different loci were treated the same way but the pathogens from adult trees were only used for phylogeny study. When clones developed well, subcultures were purified and bacteria clones were also subcultured using scaled-line culture methods. Culturing traits and clone morphology on medium were dated until a sporocarp matured.

Isolates from seedlings were further used for pathogenesis investigation using artificial inoculation. Mycelial plugs (2 mm in diameter) were applied to bark wounds of equal size on the base of the stems of seedlings of Juglans regia cv. 'Shangluo'. Controls were inoculated with sterile PDA plugs. Bacterial inoculation was also carried out by puncturing the bark with a needle. Each inoculum was applied to five stems and covered by films for 2–3 weeks, as was the control. The inoculation was carried out under controlled conditions in a chamber (temperature 25 ℃, relative humidity 75 %, and 150 μm-2 s-1 light for 16 h per day). DNA extraction, Sequencing and Blasting.

Mycelium on medium of PDA were scraped off into 1.5 mL tube and immersed into 2 % CTAB at 65 ℃, granted deliberately for few minutes and then incubated at 65 ℃ for about 1 h, followed by adding equivalent volumes of chloroform isopropanol (24:1) for proteins erasing at 8000 rpm for 10 min. The upper aqueous layer was transferred to a fresh eppendorf tube, 1/3 volume of NH4COOH (10 M) and 2.5 × volume ethanol added, and the mixture was put at -20 ℃ for 60 min. After centrifugation for 15 min at 12, 000 rpm, 4 ℃, the DNA pellet was washed with 70 % (v/v) ethanol for 2 times, and dried in a Vacuum Pump. The dried pellet was resolved in 100 μl of TE-buffer (0.01 mM Tris–HCl, pH 8.0, containing 0.1 mM EDTA), dissolved at 65 ℃ and then stored at -20 ℃ until use. Internal translate space (ITS) fragment DNA was amplified with universal primes ITS₁ and ITS₄ (White et al. 1990). PCR reactions were carried out in a total mixture volume of 25 μL containing a final concentration of 1.5 mM MgCl2, 0.5 mM dNTPs (Applied Biosystems), 10 pmol of each primer, 1.0 U AmpliTaq® DNA Polymerase (Applied Biosystems), and 20 ng of genomic DNA. Samples were incubated in a thermal cycler (PTC-100, MJ Research, USA) and followed the reaction protocol: 94 ℃ for 3 min pre-denature, 94 ℃ for 2 min denature, 53 ℃ anneal for 1 min 30 s, 72 ℃ extension for 1 min 30 s, after 10 cycles, continued as following: 93 ℃ for 1 min, 51 ℃ for 55 s and 72 ℃ for 1 min, 25 cycles later, with a final extension at 72 ℃ for 10 min 30 s, and then stop reaction at 25 ℃ for 10 min. PCR products were electrophoresis on 1.5 % agar gel (Melt tempt. 36–37 ℃), and then purified with the ZymocleanTM Gel DNA Recovery Kit (Zymo Research Corp.) by following manufacturer's instructions. Samples were then sequenced using ITS₁ and ITS₄ primers and the sequencing reaction products were cleaned following differential precipitation with ethanol. Sequences were determined by an ABI 3130 xl automated sequencer (Applied Biosystems) and deposited in GenBank (Table 1). Nucleotide BLAST search (unmodified options) was used to compare our sequences to those presented in the GenBank.

Phylogeny tree construction

DNA sequences were aligned first with Clustal X 1.81 (Thompson et al. 1997), and then adjusted based on the algorithm of Nei and Kumar (2000). All positions containing gaps and missing data were completely eliminated from the dataset. Data was analyzed by maximum parsimony (MP) using the Close-Neighbor-Interchange algorithm (Nei and Kumar 2000) as implemented in MEGA 4.0 (Tamura et al. 2007). Support for the branches based on parsimony criteria was estimated by bootstrap analysis using 1000 replicates.

Results and discussion

Fungal colonies and 2 bacterial colonies were dated and subcultured after 1 week. Sub-cultures of fungi quickly developed cottony, aerial mycelium. Colonies were initially pale to white on PDA, but diffused yellow pigment into agar around the colony 5 days later, Fig. 1b. After 10 days, aerial mycelium became grey, and submerged mycelium became dark violaceous. Conidiomata were unilocular pycnidia and conidia began to disperse from the ostioles within 10–15 days. Conidiomata were papillate, partially immersed in the medium, and covered with dark olive-green, appendage-like hyphae. Macroconidia were hyaline, thin-walled, aseptate, fusiform, with a subobtuse apex and a truncate base, Fig. 1c. One or two light yellow septa developed as spores begin matured, Fig. 1d. The mean size of 50 macroconidia was 22.3 ± 0.8 × 5.1 ± 0.3 μm; mean length/width ratios were 3.8 ± 0.1 μm. Microconidia were rod-shaped to ovoid with rounded ends and thick walls, and they measured 3–5 × 1–2 μm, Fig. 1c. Conidiophores were hyaline, unbranched, and cylindrical, Fig. 1e, f. On walnut stems, conidiomata were dark grey to black, eustromatic, separate, uni-or multilocular. Locules were up to 200 μm in diameter, immersed in the bark, and sub-epidermal. Conidiophores were the same as that in PDA medium but longer, 7.3–21.5 × 4–6 μm, with 1–3 indistinct percurrent proliferations, Fig. 1f. Conidia were similar shape to those produced in PDA medium but more variable in size, and ranged from 12–25.5 × 4.5–8.2 μm, with a mean length/width ratio of 2.75 ± 0.6 (100 conidia). Bacteria clone were identified as Bacillus subtilis by 16SrDNA (accession no.KF591602-KF591603) and were reported in another paper (Yu et al. 2015).

Inoculation experiment showed the putative pathogen can infect via wounded bark. After 2–3 weeks, all seedlings inoculated with the putative fungi pathogen showed the expected symptoms, with cankers and copious exudates at inoculation sites, Fig. 2. Symptoms were not visible in the control or in the stems inoculated with bacteria. No fungus was reisolated from controls. The putative pathogen was re-isolated from all stems.

Based on the symptoms, cultural morphologies, and conidial characters, the fungus was tentatively identified as Neofusicoccum parvum according to (Slippers et al. 2004). Neofusicoccum parvum has previously been reported as causing canker symptoms of Vitis vinifera in Spain (Martos et al. 2011; Moral et al. 2010), and Mangifera indica in Australia (Slippers et al. 2005), and Eucalyptus in Uruguay (Perez et al. 2010), and walnut in California (Michalidis et al. 2012). It was reported occurring on Populus, Vitis and Eucalyptus (Yu et al. 2009) in recent years in China, and to our knowledge, it is the first report of N. parvum as a canker pathogen of seedlings of Juglans regia in China. The isolates used in this study are maintained in the culture collection of the Forestry College, Northwest A & F University, China, as culture collection number NW11-016 and NW11-014.

However, N. luteum, B. dothidea and N. ribis are similar to N. parvum. Pennycook and Samuels (1985) described N. luteum and separated it from N. parvum and B. dothidea according to the yellow pigment diffused in young PDA culture. Phillips et al. (2002) indicated that this is not a reliable character unless the cultural conditions are controlled carefully. Instead, they described the unilocular conidiomata of N. luteum as distinct from N. parvum and B. dothidea. However, this is also influenced by culturing conditions. (Slippers et al. 2005) found N. ribis and N. parvum both diffused yellow pigment in young culture and suggested conidium dimensions and aerial mycelium of aged colonies as being important characters to distinguish N. parvum, N. luteum, N. ribis and B. dothidea.

To determine the systematic placement of the putative pathogen in this paper, the ITS rDNA locus was amplified and sequenced with the universal primers ITS₁ and ITS₄ (White et al. 1990), followed by a BLAST search in GenBank and a phylogenetic study, Table 1.

Sequences of the putative pathogen (NW11-016) and a similar culture (NW11-014) from cankered seedlings showed 100 % sequence identity with N. parvum (Fig. 3). All isolates (Table 1) from adult stems and branches of Juglans regia were in the B. dothidea clade, which is consistent with the report of Tang et al. (2012). B. dothidea was thought a complex species (Slippers et al. 2004; Pennycook and Samuels 1985), and can be found in a wide host range (Smith 1934). There were many cryptic species associated with B. dothidea, now it can be distinguished by help of molecular technologies (Slippers et al. 2004; Denman et al. 2003). N. luteum in this analysis was monophyletic. N. ribis and N. parvum are clearly closely related (Sakalidis et al. 2011) and this was confirmed here, and it is confusing to find both N. ribis (Urbez-Torres et al. 2012) and N. parvum (Martos et al. 2011) ever described as canker pathogen of Vitis vinfera.

It is also important to note that N. ribis has been reported as a walnut pathogen (Rumbos 1987), and B. ribis (anamorph: N. ribis) was ever thought as a synonym of B. dothidea (Michailidis 1991; Whitch and Clayton 1963). Since most Botryspaheria species are cryptic, other coding sequence genes may be useful for further identification (Inderbitzin et al. 2010). In this paper, we reported this seedling pathogen was Neofusicoccum parvum newly occurred on walnut tree.

Acknowledgments

I would thank deeply George Newcombe in University of Idaho for his English reviewing and suggestion. This study was supported by the program for tacking key problem of Shaanxi agricultural scientific and technological extent (2015NY124), Project NSFC(31270690), Project PCSIRT (NO. IRT1035), special funding for basic s & t work of Ministry of Science and Technology (2009FY210100) of China.