New record of boxwood volutella blight fungal agents in Iran

Morphological and molecular studies

In order to identify fungal species, macroscopic and microscopic characteristics were analyzed. The fungal structures were mounted on slides in 50 % Lactic acid and examined with the Olympus BH2 microscope. Microscopic images were provided by a Leica microscope equipped with a Canon digital camera. For measurement of fungal structures, at least 30 structures in each case were measured (Bezerra 1963, Rossman et al. 1993, Lombard et al. 2015).

For the molecular study, genomic DNA was extracted from mycelia grown on PDA using 5 % Chelex (Hirata and Takamatsu 1966; Walsh et al. 1991). The internal transcribed spacers (ITS1, ITS2 and 5.8S) regions were amplified using primers ITS1 (5'- TCC GTA GGT GAA CCT GCG G -3') and ITS4 (5'- TCC TCC GCT TAT TGA TAT GC -3') (White et al. 1990). The PCR product of each isolate was submitted to Bioneer Company (South Korea) for sequencing. All the sequences were edited in MEGA7.0 (Kumar et al. 2016) and compared with sequences available at the GenBank nucleotide database using a BLAST search (Altschul et al. 1990). Several reliable sequences with high similarity to our sequences were selected for phylogenetic analysis. The phylogenetic tree was inferred using the Minimum Evolution method in MEGA7.0 (Kumar et al. 2016). The evolutionary distances were computed using the Kimura 2-parameter method. The rate variation among sites was modeled with a gamma distribution (shape parameter =1). The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. All ambiguous positions were removed for each sequence pair. There was a total of 549 positions in the final dataset.

Pathogenicity tests

The inoculum preparation was conducted based on the Shi and Hsiang (2014b) protocol. Seven days after incubation of P. buxi and P. foliicola on PDA at 25 °C, the colonies were washed with sterilized water and the spore suspension with mycelia was passed through multilayer sterilized gauze. Subsequently, a suspension of 1×10⁶ (spores/mL) was used for inoculation. For the pathogenicity test on detached leaves, the leaves were disinfected for one minute with 70 % ethanol, then washed three times with sterile distilled water. They were then immersed in spore suspension for one minute. The number of four detached leaves was placed in 8-cm-diameter Petri dishes lined with filter paper. Two milliliters of sterile distilled water were added to each Petri dish to maintain high humidity during the test. All of the cultures were sealed with parafilm and placed at 25 °C under a 12:12 light-dark regime. These experiments were repeated at least two times. Sterile distilled water was used for negative control. The leaves were observed after 7 days.

Additionally, two-year-old healthy potted plants of Buxus sempervirens subsp. hyrcana were used for pathogenicity test according to Garibaldi et al. (2016). Some leaves of healthy plants were cut about by half before the inoculation and whole bushes inoculated by spraying with a spore suspension of 1×10⁶ (spores/ml) concentration. Control plants were inoculated with sterile water, then were covered with plastic bags for 5 days and maintained in a growth chamber at 25±4 °C (Fig 3). Following inoculation, the pathogenicity of two isolates on detached leaves and progress of symptoms were scored according to Table 1.

Table 1. Scoring the virulence of two Pseudonectria species isolates on Buxus sempervirens subsp. hyrcana on detached leaves and greenhouse experiments.

The disease severity index (DSI) was calculated based on the following formula (Wheeler 1969):

DSI =  (∑ (Number of leaves×Disease score) /(Number of leaves observed×Maximum disease score)

Statistical analysis was made for comparison of pathogenicity of Pseudonectria species on the detached leaves and plants using disease severity. The analysis was performed on the Arcsine transformations of severity values using Excel 2016 software. Comparisons of the means were performed by Student’s t-test using Tukey test at a probability level of (P<0·01) using SAS® software. The Microsoft Excel software (2016) was used to draw plot charts.

RESULTS AND DISCUSSION

Morphological and molecular identification

According to morphological and molecular studies, two species including P. buxi and P. foliicola were identified. Voucher specimens are deposited in the mycology herbarium of the University of Guilan (GUM).

Fig 3. A Minimum Evolution tree based on ITS sequences for 44 sequences of Pseudonectria and some related fungi from Hypocreales. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method. All ambiguous positions were removed for each sequence pair. The numbers above the branches represent branch support using 1000 bootstrap replications (Bootstrap values below 50 % are not shown). Evolutionary analyses were conducted in MEGA7.0.

Table 2. Mean comparison of the disease severity on detached leaves in Pseudonectria species by t-test.

Table 3. Mean comparison of the disease severity in greenhouse conditions in Pseudonectriaspecies by t-test.

The difference between the performance of the two species on detached leaves was obtained 38.1, which was statistically significant (diff=-38.1; t=-3.95; P=0.009).

The difference between the performance of the two fungi under greenhouse conditions was obtained 54.05, which was statistically significant (diff=-54.05; t=-7.01; P=0.0.0001).

As shown in this study, both species could not cause disease on the healthy leaves of the plant, but they can cause disease only in the presence of wounds on the plant. During recent years, boxwood tree in the north of Iran has threatened by two major pests including the invasive fungus C. pseudonaviculata and box tree moth (Cydalima perspectalis) and considerable natural scope were destroyed and disappeared. Although Pseudonectria species penetrate the plant through the wounds, such wounds are usually available in nature as a point of infection for these fungi. When the fungus enters the leaves, disease development is much faster than one may expect, the leaves begin to yellow and detach from branches. Unfortunately, under humid conditions, which is available in northern Iran, the fungi further grow and produce abundant sporodochia on fallen leaves, therefore, the whole surface of leaves is covered by the fungus reproductive structures. Hence, occurring of these fungi along with the two above-mentioned pests on box trees can promote death and destruction of the scope of the trees in the northern forests of Iran.

Fig. 4. Pathogenicity test of two Pseudonectria species a. and c. symptoms produced by Pseudonectria buxi. b. and d. symptoms created by Pseudonectria foliicola.

Acknowledgment

This work was supported by a grant from the Deputy of Research and Technology of the University of Guilan, Rasht, Iran.