One of the main culprits of Morchella ( Morohellaceae : Morchella ) pileus rot disease : Pestalotiopsis trachicarpicola


 We found some diseased fruiting bodies when cultivating Morchella. The infected ascomata were covered by white mycelia mainly on the pileus, and the infection cause the fruiting body to rot and die. A total of 5 fungi were separated from symptomatic tissue. One of them was proved to be pathogenic by Koch’s postulates. The pathogen was identified as Pestalotiopsis trachicarpicola based on its morphology and Multi-gene (ITS,β-tubulin and tef1) analysis. This is the first report of pileus rot disease on cultivated Morchella caused by P. trachicarpicola in China, which is of positive significance for the quarantine of cultivated soil of Morchella.


Introduction
Morchella is a kind of edible and medicinal mushrooms with high economic and scienti c research value  (Zhang YC et al., 2020), these might be only a part of Morel pathogenic fungi, and more remain to be discovered.
During the cultivation of M. sextelata, we found a very harmful fungal disease, which caused huge economic losses to the farmers. We speculate that the pathogen may come from the soil, in the early development of M. sextelata, it attached to the fruiting body cap, as M. sextelata grows, its mycelia gradually invade the interior of the ascomata pileus, eventually make whole fruit body rot and die, result in a serious decline in the quality and yield of M. sextelata. We isolated a pathogenic fungi from this nidus, through morphological identi cation and Multi-gene (ITS,β-tubulin and tef1) analysis, it was identi ed as Pestalotiopsis trachicarpicola.

Pathogen isolation
The infected M. sextelata was placed in Clean Bench and the surface of fruiting body was washed with sterile water, then cut the diseased plaque and inoculated on potato-dextrose agar (PDA) medium, cultured at 25 ℃ under dark conditions. The marginal mycelia were picked for isolation of pure culture, when the mycelia began to germinate. The puri ed pathogen strain is stored at 4 ℃ .

Pathogenicity tests
Koch's postulates was used to verify the pathogenicity of pathogen. Firstly, healthy ascomata were selected in the cultivation eld of M. sextelata and a 5 mm hole was inserted on the cap with sterile toothpick. Meanwhile, conidia were cultured on PDA plate and eluted to prepare suspension. Then, 20µL suspension was inoculated into the hole and sterile water was used as negative control.
After the fruiting body of M. sextelata showed symptoms, isolation of pure culture was carried out again from the diseased spot, and if the pure culture was completely consistent with the previous culture, the fungus was identi ed to be pathogen.

Morphological observation
The pathogen was inoculated on PDA plate and cultured at 25°C in Darkness for 3-15 days to visually observe the color, diameter and characteristics of hyphae. Approximately 25 random conidia were examined for morphological characters (the size, the shape, color type, the number and length of apical and basal appendages) using Optical Microscope. Identi cation was based on the keys and

DNA extraction, PCR ampli cation and nucleotide sequencing
The pathogenic fungi were grown on PDA plate and cultured at 25°C in Darkness for 9 days. The fresh mycelium was scraped from the surface of the culture medium, and the whole genomic DNA of the pathogen was extracted with a fungal DNA extraction Kit(sangon Biotech, shanghai). The internal transcribed spacer regions, interval 5.8 S nrRNA gene (ITS) was ampli ed using primer pairs ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)and ITS4 5′-TCCTCCGCTTATTGATATGC-3′ (White TJ et  PCR was performed in a 25µL reaction system containing 10.3µL of double distilled water, 12.5µL 2×Taq MasterMix(sangon Biotech,shanghai),0.6µl of each primer (10µM) and 1µL DNA template. The thermal cycling program was as follows: For ITS an initial denaturing step of 95°C for 3 min, followed by 30 ampli cation cycles of 95°C for 30 s, 52°C for 45 s and 72°C for 90 s, and a nal extension step of 72°C for 10 min. For β-tubulin PCR conditions were an initial step of 3 min at 95°C, 30 cycles of 30 s at 95°C, 50 s at 55°C, and 1 min at 72°C, followed by 10 min at 72°C. For tef1, an initial step of 3 min at 95°C,30 cycles of 30 s at 95°C, 55 s at 58°C, 90 s at 72°C (Maharachchikumbura SSN et al., 2012). The PCR products were detected by 1% agarose gel electrophoresis and sent to Sangon Biotech for sequencing.

Phylogenetic analysis
The sequencing results were compared and analyzed in NCBI database by Blast. Similar strains were selected for multi gene joint tree construction. Combination of multi-locus dataset of three gene regions was phylogenetic analyzed by using MEGA7 software (Kumar S et al., 2016). The sequence alignments algorithm adopts ClustalW to delete the unaligned sequence manually after the alignment, and then uses the method of Maximum Likelihood (ML) to build the phylogenetic trees. The branch support of ML analysis was evaluated by 1,000 bootstrap replications.

Pathogen isolation and Pathogenicity tests
In this study, ve strains of fungi were isolated from the fruiting body of diseased M. sextelata and numbered M-1, M-2, M-3, M-4 and M-5 respectively. It was found that the fruiting body of M. sextelata inoculated with M-5 appear obvious lesions by Koch's postulates veri cation (Fig. 1b), and the symptom of the lesions was consistent with that of the naturally diseased ascomata (Fig. 1a), however, there was no symptom in the control group (Fig. 1c), this showed the fungus M-5 really was a Morchella pathogen.

Phylogenic study
The PCR ampli ed fragments were sent to the company for sequencing to obtain the partial sequence of ITS of 586bp, the partial sequence of β-tubulin gene of 458bp and the partial sequence of tef1 gene of 965bp. The three sequences were uploaded to GenBank and the accession numbers were OL362082, OL828342 and OL905009, respectively. The sequencing results were compared on the NCBI website, and the results showed that the three sequences of M-5 strain were identical with Pestalotiopsis trachicarpicola, homology is as high as 99% − 100%. The phylogenetic tree was constructed by using MEGA7 software, and the information of the tree was shown in Table 1. The pathogen M-5 strain and Pestalotiopsis trachicarpicola strain YB1 were clustered in the same branch, and the test value was as high as 100% (Fig. 3).
The pathogen M-5 was identi ed as Pestalotiopsis trachicarpicola by morphological identi cation and multi-gene analysis.

Discussion
The pathogen of M. sextelata is classi ed in Pestalotiopsis spp.. This genus, is characterized by ve cells and the middle three by dark color, which is a fungi imperfecti and was established and named by Steyaert in 1949(Steyaert RL, 1949. It is very di cult to classify and identify (Maharachchikumbura SSN et al., 2011). At present, the morphology of conidia is the most widely used taxonomic feature of Pestalotiopsis spp., which is classi ed by the length, width, cell color, number and length of attached laments of conidia (Jeewon R et al., 2004).However, because of the morphological similarity between species, only observing the morphology of conidia is not enough to accurately identify the species. Therefore, the molecular data are also gradually used in the classi cation of this genus, and the classi cation and identi cation system of this genus is gradually improved from single analysis of conserved ITS sequence to joint analysis of multiple genes (Liu AR et al., 2010). Now, the most commonly used multi-gene joint analysis is through the combination of three sequences: ITS sequence, tub2 and tef1, which shows better differences among different species (Maharachchikumbura SSN et al., 2012). In this study, the pathogen of M. sextelata was identi ed as Pestalotiopsis trachicarpicola by the method described above.
Pestalotiopsis spp. is an important plant pathogen, which causes diseases all over the world. It can not only cause diseases of seeds and seedlings, but also cause leaf spot, branch and trunk blight and fruit rot. Among them, leaf spot is the most common, and the main host includes Camellia sinensis (Maharachchikumbura SSN et  . Initially, the necrotic lesions appeared on the branch and trunk, which led to the discoloration and cracking of the bark with the development of the disease, branch and trunk with the lesion were wilt at the end. Fruit rot mainly occurs in the harvest and storage period of Grape (Ghuffar S et al., 2018, Jayawardena RS et al., 2015, kiwifruit (Li L et al., 2016), rambutan (Keith LM, 2008), Guava (Keith LM et al., 2006), Persimmon (Palou L et al., 2015)etc. Dark brown to black spots appears in the disease spot at early stage and white hyphae grow in the later stage, whole fruit rots at the end. If the fruit is damaged, it is more likely to be infected. The infection phenomenon of each species will be different. In addition, it also causes the problems of Cunninghamia lanceolata (Zeng SH, 1998, Huang TZ, 1983, strawberry (Hua XF et al., 2021) rotten seed, rotten root and seedling growth decline. However, so far, the pathogenic effect of this genus on macrofungi has not been found.
It has been reported that the Pestalotiopsis spp. mainly take the soil, diseased seedlings and organization as the transmission carriers, which prefer to grow in the high humidity environment. The younger the infected tissue is more likely to get infected and tissue damage is also conducive to pathogen infection (Ge QX et al., 2009). Morchella is suited to grow in high humidity environment, and its tissue is very fragile and vulnerable to mechanical and insect damage, which increase the possibility of Morchella infection (Ruíz-Herrera J and Osorio E, 1974, Sambyal K and Singh RV, 2021). Most importantly, as an edible commercial mushroom, Morchella grows and is harvested in a short time (Zhi CY et al., 2021). Once infected, it loses all commercial value, therefore, we should pay more attention to the soil quarantine work in the cultivation process of Morchella.

Declarations
Informed consent statement All authors have seen a copy of the manuscript and have approved its submission.
Author contribution LHL conceived the idea and designed the experiments. ZL carried out the research experiments and analyzed the results. ZL drafted the manuscript. LHL revised the manuscript.

Data Availability Statement
Some or all data, models, or code generated or used during the study are available from the corresponding author by request. (List items). Table   Table 1 is only available as a download in the Supplemental Files section.