Identification of Fusarium solani f. sp. cucurbitae Causing Zucchini Fruit Rot in Inner Mongolia, China

Abstract

Fruit rot is one of the major diseases impacting the production and quality of zucchini (Cucurbita pepo). In August 2021, fruit rot symptoms were observed on the zucchini fruit ‘Jindi 1’ in the Wuyuan region in Inner Mongolia, China with an incidence ranging from 10% to 30%. Where the pepo was in contact with the soil, dark grey and spongy corky lesions 4–5 cm in diameter with a light brown halo were observed. The internal necrosis of the fruit rind was also recorded. From the affected fruits, fungal colonies belonging to the Fusarium species were exclusively isolated. Molecular analysis of the ITS, TEF-1α, and RPB2 sequences identified the isolates as Fusarium solani f. sp. cucurbitae. Inoculated on ‘Jindi 1’, the strain Fx-1a induced typical fruit rot on the pepo and wilting on seedlings, while negative-controls remained asymptomatic. The impact of this disease on seed quality and yield in zucchini seed production needs to be further studied.

1. Introduction

Zucchini (Cucurbita pepo), a highly polymorphic vegetable crop, is an economically important crop in the Cucurbitaceae family worldwide [1]. Zucchini is widely cropped for seed production (three to four hundred seeds per pepo) in more than twelve provinces in China, including Gansu, Xinjiang, Heilongjiang, Shanxi, and Inner Mongolia [2]. Given their health benefits and economic value, zucchini seeds have received increasing attention, and have become important economic crops in the north of China [3]. As a result, the production area in 2016 was about 87,000 ha in Inner Mongolia and more than 200,000 ha in China (unpublished data). Given the economic value of the seed-producing zucchini crop, understanding potential diseases is necessary.

Fusarium foot and fruit rot in pumpkin, caused by F. solani f. sp. Cucurbitae, was widespread within the United States during 2001 to 2003 [4]. The pathogen has also been determined as the first reported fungal disease affecting cucurbits production in the Almerίa province of Spain [5] and in Arkansas state in the United States [6]. Recently, some studies have investigated fungal diseases on plants in the Cucurbitaceae family including fruit rot of melon caused by Fusarium asiaticum [7]; Cucurbitaceae root rot caused by F. solani in Zhejiang Province in China [8]; a gene from F. oxysporum f. sp. melonis (Fom), which determined the difference in host range compared with F. oxysoporum f. sp. radicis-cucumerinum (Forc) [9]; and the pathogenicity of five species of Fusarium (Fusarium sp., F. sulawesiense, F. falciforme, F. kalimantanense, and F. pernambucanum) and their effects on melon fruit quality in Brazil [10].

In the harvest season of seed-producing zucchini (August 2021), symptoms of fruit rot were observed on ‘Jindi 1’ (Figure 1a), a commercial cultivar used for seed production (Figure 1b), in the Wuyuan region of Inner Mongolia with an incidence ranging from 10% to 30%. The affected fruits showed dry, dark grey, spongy lesions (4 to 5 cm in diameter) with a light brown halo (Figure 1c,d). The typical symptoms occurred where the fruit was in contact with the soil, which was not easily noticed by farmers. The internal necrosis of the fruit rind was also observed (Figure 1e,f). In this study, F. solani f. sp. cucurbitae was isolated from the infected pepo and identified by morphological features and molecular tools. Pathogenicity tests were also performed to confirm the aetiology of the observed symptoms.

2. Materials and Methods

2.1. Samples Collection

Six infected fruits were collected from three different fields for pathogen isolation and identification.

2.2. Isolation and Purification of the Pathogens

Portions (0.5 × 0.5 × 0.5 cm in size) were cut out from the surface of symptomatic fruit and disinfected by a succession of treatments: 1 min in 2% NaClO and 1 min in 75% ethanol followed by 2 min rinses in sterile water [11]. Disinfected material was dried on sterile filter paper and placed on petri dishes containing potato dextrose agar (PDA). After incubation at 25 °C in the dark for 5 days [12], developed fungal cultures were purified using the single spore method [13].

2.3. Morphological and Molecular Identification

Morphological characteristics were observed by culturing the isolates on PDA at 25 °C in the dark for 5 days. The spore morphology of the isolates were observed by using a compound microscope (ECLIPSE NI-U^®, Nikon, Tokyo, Japan) fitted with a digital camera (Y-TV55^®, Nikon, Tokyo, Japan).

For the total DNA extraction, isolates were cultured in Potato Dextrose Broth (PDB) at 25 °C on a shaker at 150 rpm for 5 days [14]. Genomic DNA was extracted using the EasyPure^® Genomic DNA Kit (TransGen, Beijing, China) according to the manufacturer’s instructions. The internal transcribed spacer region (ITS) of isolates was amplified by using the primer ITS1(5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The part of the translation gene (TEF-1α) and the second largest subunit of the RNA polymerase II gene (RPB2) were amplified by using the primers EF1 (5′-ATGGGTAAGGAGGACAAGAC-3′) and EF2 (5′-GGAAGTACCAGTGATCATGTT-3′) and using the primers 5f2 (5′-GGGGWGAYCAGAAGAAGGC-3′) and 7cr (5′-CCCATRGCTTGTYYRCCCAT-3′), respectively, under the PCR reaction conditions described previously [5]. 2× EasyTaq^® PCR SuperMix (TransGen) was applied and the PCR reaction mix was assembled into 50 µL volume. The amplicons were Sanger sequenced at BGI Tech Solutions (Beijing Liuhe) Co., Limited, Beijing, China. The TEF-1α and RPB2 gene sequences selected from NCBI and the Fusarium database [15] were analyzed and combined in Geneious and aligned by Clustal W and analyzed using maximum likelihood method with bootstrap replications (=1000) in MEGA 11.0.

2.4. Pathogenicity Test

Pathogenicity tests were performed twice independently to evaluate the pathogenicity of the isolate on zucchini fruits. The ‘Jindi 1’ fruit were washed under running water, their surfaces were disinfected by spraying them with 75% ethanol 3 times, and each treatment was about 5 min [5]. Six wounds (approximately 1–2 mm deep) were made with a sterile 5 mm diameter cork borer on the exocarp. Four mycelial plugs (5 mm) cut from the margins of a 8-day-old culture on a PDA plate were placed mycelium side down into the wounds, and two plain PDA plugs were inoculated as controls [5,16]. Symptom development was assessed up to 9 days post inoculation (dpi). The pathogenicity of the Fx-1a isolate was also assessed on zucchini seedlings at the first true leaf stage grown in sterile plastic pots (10.5 cm × 8.8 cm, one plant per pot) [5,17] filled with sterile commercial potting mixes containing peat, vermiculite, and pearlite. A 10-day-old colony on PDA was scraped and suspended in distilled water to obtain a 10⁶ CFU/mL suspension. Aliquots (4 mL) were applied in the soil around the stem of each plant without [4]. Six replicates were performed. Four seedlings served as uninoculated controls. Fruits and seedlings were kept at 23 ± 1 °C in a humid chamber under 16 h light/8 h dark and 65 ± 5% of relative humidity [6].

Re-isolation of the pathogen was conducted while the artificially inoculated fruits and plants developed symptoms according to the isolation methods described above (Section 2.2). Then the reisolated fungi were identified by morphological and molecular characteristics to complete Koch’s postulates.

3. Results

3.1. Morphological Identification

Colonies with a white to straw, cottony mycelium were exclusively isolated from symptomatic fruits. Aerial mycelium formed long and simple or branched conidiophores which produced abundant zero- to one-septate microconidia (7.1 × 2.4 μm to 19.1 × 3.6 μm) and 3–4 septate macroconidia (40.5 × 4.8 μm to 67.0 × 6.0 μm). Intercalary chlamydospores were also produced (Figure 2). Ten isolates from symptomatic fruits were consistently identified as Fusarium species based on their microconidia and macroconidia characters [18] in PDA.

3.2. Molecular Identification

The amplification of the 16S rDNA region generated a fragment of 534 bp (NCBI Accession Nos.: OL871487 and OL871488 for Fx-1a and Fx-1b, respectively). The TEF-1α amplification generated a fragment of 718 bp (NCBI Accession Nos.: OL870950 and OL870951 for Fx-1a and Fx-1b, respectively). The RPB2 gene amplification generated a fragment of 879 bp (NCBI Accession Nos.: ON631223 and ON631224 for Fx-1a and Fx-1b, respectively). The amplified fragments had 100% identity homology with those of F. solani, e.g., MT032588 (ITS), KF372878 (TEF-1α), and OK595060 (RPB2) in the NCBI GenBank database.

According to TEF-1α and RPB2 phylogenetic analysis (Figure 3), Fx-1a and Fx-1b clustered together with the F. solani f. sp. cucurbitae sequences (AF178327 and EU329489).

3.3. Pathogenicity Test

The two selected isolates reproduced the symptoms observed in the field; lesions over 5 cm in diameter were observed on ‘Jindi 1’ fruits at 9 dpi (Figure 4a–c).

All inoculated seedlings exhibited brown, water-soaked rot at the base of the stem with chlorosis and wilting at 12 dpi (Figure 4d–f), whereas no symptoms were observed on the plant used as control (Figure 4d).

Fungal cultures re-isolated from artificially inoculated materials exhibited the same morphological traits as the original isolates used for inoculation, thus fulfilling Koch’s postulates.

4. Discussion

The fungal diseases of plants in the family Cucurbitaceae mainly include Phytophthora blight caused by Phytophthora capsici, powdery mildew caused by Podosphaera xanthii, and Fusarium diseases caused by Fusarium species [19,20]. Fusarium fruit rot is an important disease affecting melons and pumpkins, which leads to economic and quality losses [6,10]. Recent studies revealed F. asiaticum and F. incarnatum, as novel pathogens, can cause fruit rot on melons in China [7,21,22], while F. solani f. sp. cucurbitae that was associated with Fusarium fruit rot on zucchini was not addressed anywhere in China. This work demonstrated that the causal agent of fruit rot in seed-producing zucchini was F. solani f. sp. cucurbitae in the Wuyuan region of Inner Mongolia. It also provided information on the diagnosis and identification of fruit rot in seed-producing zucchini. To our knowledge, this was the first report of fruit rot in zucchini caused by F. solani f. sp. cucurbitae in Inner Mongolia, China.

The pathogen F. solani f. sp. cucurbitae was firstly demonstrated to spread by seed transmission in 1961 [23]. Previous studies showed that seed infestation and infection occurs once the lesion (caused by F. solani f. sp. cucurbitae) on the fruit rind extends to the margins of the seed cavity [24]. In this study, it was observed that the lesions on the infected zucchini fruit developed inside the exocarp in fields, so did it during pathogenicity assays. Based on this knowledge, it was speculated that the disease observed in this work might reach the seed cavity and cause the infection of the seeds. Given that Fusarium species can survive in seeds but does not affect germination or viability [24], the seed borne transmission of fruit rot in seed-producing zucchini is possible, which we are presently investigating further. We, however, address the seed industry, calling attention to the potential risk of the F. solani f. sp. cucurbitae to limit the spread of contaminated seeds.

In addition, we observed that the F. solani f. sp. cucurbitae obtained in this work caused rot symptoms on both the zucchini seedlings and on the fruits by artificial inoculation. It was consistent with previous reports on this pathogen, which is a causal agent of Fusarium crown and root rot [4,25]. Therefore, the occurrence of the root rot caused by F. solani f. sp. cucurbitae needs to be monitored further, even though the symptoms of the disease of rot on roots were not obvious in the fields of Wuyuan. As reported, F. solani f. sp. cucurbitae can survive for at least 20 months under greenhouse conditions [25]; managements to control the pathogen where the fruit rot or root rot is detected are necessary.

Fungicide application is one of the effective treatments to inhibit F. solani f. sp. cucurbitae, including carbendazim, prochloraz, thiophanate-methyl, and hymexazol [8,25]. It is recommended to apply fungicides prior to the development of the disease. Given that the fruit rot observed on seed-producing zucchini in this work commonly occurred where the fruit was in contact with the soil, it may be easily ignored by farmers in its early stages. Therefore, the timing of fungicide application is more important. In addition, the host range of F. solani f. sp. cucurbitae isolated from zucchini plants was described and reported by previous studies; it could infect watermelon, gourd, melon, cucumber, and pumpkin, excluding Solanaceous vegetables and cruciferous vegetables [5,26]. Hence, crop rotation could be another feasible approach to decrease the disease based on the information of the pathogenic range of the pathogen.

In summary, although the impact of Fusarium fruit rot caused by F. solani f. sp. cucurbitae on the yield and seed quality of seed-producing zucchini is still under investigation, it was urgent to draw more attention to this disease so that more information on the identification and management of it in fields could be provided.