Introduction

Sweet potato (Ipomoea batatas (L) Lam.) is a widely cultivated food root of great economic importance due to its widespread adaptation to the most diverse regions, climates and soils (Queiroga et al. 2007). Although it is produced and consumed throughout the country, several factors limit its production, among them, root rot caused by fungi. The fungi that cause this disease have the ability to remain in infected plant residues for long periods, favoring the spread of the disease to other areas of cultivation. Most soil-dwelling pathogens have a high competitive capacity and remain in the soil for a long time, even after long periods of crop rotation (Michereff et al. 2005). To date, sweet potato root rot is associated with several fungal species including: Rhizopus sp., Lasiodiplodia theobromae, Ceratocystis fimbriata, Diaporthe destruens (= Phomopsis destruens, Plenodomus destruens), D. kongii, and Fusarium solani (Boerema et al. 1996; Pio-Ribeiro et al. 2016) and root and stem rot caused by Macrophomina, Lasiodiplodia and Neoscytalidium belonging to the Botryosphaeriaceae family (De Mello et al. 2021).

The species Geotrichum candidum, previously known as Galactomyces candidum, is a fungus with great phytopathological importance. Some species are important post-harvest plant pathogens of fruits and vegetables that cause economic losses in agriculture worldwide, due to the occurrence of sour rot in stored fruits and vegetables. These fungi infect fruits and vegetables through injuries caused by insects or mechanical wounds mainly in the storage stage (Agrios 2005).

According to the study Kim et al. (2022), root rot in sweet potatoes treated with soil containing F. solani and F. oxysporum before the storage period was investigated, and the difference in the incidences of root rot of sweet potato among three fields with similar storage conditions was confirmed. These results suggest that the characteristics of the cultivation soil are important for the incidence of root rot of sweet potato.

Material and methods

Between the years of 2017–2019, in a sampling of a study of Mello et al. (2021) in 2017 and of a study in progress (sampling in 2019, de Mello et al., in prep.), sweet potato tuberous roots with post-harvest rot were collected in private and public markets located in the municipalities of Olinda and Igarassu in Pernambuco. The samples were sent to Laboratório de Micologia Ambiental of Departamento de Micologia of Universidade Federal de Pernambuco. Indirect isolation of the fungi was carried out following the methodology used by de Mello et al. (2021). For morphological identification, the cultures were incubated in PDA at 25℃ for seven days in the dark. Microscope slides were made in lactoglycerol and thirty measurements of fungi structures were performed for species identification.

DNA extraction of the fungi isolates was performed following the methodology used by de Mello et al. (2021). The primers ITS5 (White et al. 1990) and LR6 (Vilgalys & Hester 1990) were used to amplify the rDNA-ITS region and the D1/D2 domain of the large subunit rRNA gene. Following cycling according to the work of Paes et al. (2022) the PCR products were purified and sequenced. For phylogenetic analysis, consensus sequences were compared and aligned according to the GenBank database using the BLASTn program. Bayesian Inference (BI) analysis was performed applying the Markov Monte Carlo chain method (MCMC), being concluded with Mr. Bayes v.3.1.1 (Ronquist and Huelsenbeck 2003) on the CIPRES portal (Miller et al. 2010) with 10,000,000 generations. The HKY + G nucleotide substitution model was generated for the LSU region, being estimated with the Akaike Information Criterion (AIC).

The pathogenicity test was performed following the methodology described by Holmes & Clark (2022). Sterile toothpicks were dragged through pure cultures of three G. candidum isolates and inserted (1.5 cm deep) into the midsection of sweet potato roots (cv. canadense). The roots were submerged in sterile distilled water and incubated at room temperature for 5 days. Five roots were used per treatment. For the control, the toothpicks were scraped in sterile PDA.

Results

Several root samples showed soft necrotic lesions and the presence of white mycelium under the lesions presenting a strong odor. Six isolates of Geotrichum were obtained from tuberous roots samples (three isolates from Olinda and three from Igarassu) in different years and one isolate, obtained from one sample of sweet potato stem rot, collected in municipality of Aliança, Pernambuco, in 2019 of another study in progress (de Mello et al., in prep.), was included in the analyses.

The phylogenetic tree obtained by Bayesian inference of LSU gene region showed that the DNA sequences of seven isolates (GenBank Acc. Nos. ON753543 to ON753549) grouped in a clade with Geotrichum candidum sequences (Fig. 1) obtained from previous studies and with Geotrichum candidum CBS 178.71 (ex-type) (Acc. No JN974262.1).

Fig. 1
figure 1

Phylogenetic tree inferred from Bayesian analysis based on nucleotide sequences of the D1/D2 region in the 26S rRNA genes for Geotrichum species. The posterior probabilities are indicated above the nodes. The tree was rooted to Dipodascus tetrasporeus U40081. The species obtained in this study are highlighted in bold

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In the morphological analyses, the isolates showed abundantly white, dry and powdery mycelium. The hyphae measuring 2.5–5.5 µm, with early disarticulation into abundant, hyaline cubic arthroconidia measuring 5.0–12.5 × 2.5–5 µm (Fig. 2). The morphological characteristics were similar to those described for G. candidum (Carmichael 1957) confirming the identification. Three representative isolates (URM 8539, URM 8540 and URM 8541) were deposited in the culture collection “Micoteca URM” at the Universidade Federal de Pernambuco (Recife, Brazil).

Fig. 2
figure 2

Morphological aspects of the species Geotrichum candidum on potato dextrose agar (PDA) a Colony on PDA with seven days of growth; b reverse colony on potato dextrose agar; c conidia and hyphae on PDA; d conidia and hyphae on PDA; Scale bars: c = 10 µm and d = 50 µm

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In the pathogenicity test, cross section of roots was performed, the diseased tissue around the wound was darkened and soft with slightly sunken lesions (Fig. 3). None of the roots used for control showed signs of deterioration. The fungi were reisolated from tissues with rot symptoms, confirming Koch's postulates.

Fig. 3
figure 3

Pathogenicity test of Geotrichum candidum in tuberous roots of sweet potato. Negative control tuberous roots without symptoms (a-b), Tuberous roots with diseased tissue around the wound darkened and soft with slightly sunken lesions 5 days after inoculations with Geotrichum candidum isolate ARM 329 (URM 8539) (c–d); G. candidum isolate ARM 648 (URM 8541) (e–f) and G. candidum isolate ARM 650 (URM 8540) (g-h)

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Discussion

The present work provides information about the morphology, phylogeny and pathogenicity of isolates belonging to the species Geotrichum candidum associated with sweet potato sour rot in Brazil.

Similar to what happens with several species of fungi, the taxonomy of Geotrichum species based on morphology presents limitations mainly due to the existence of cryptic species that have morphological characters that overlap, making it difficult to correctly identify these species using only the morphological aspects in the identification (Hoog & Smith 2004).

Given this context, carrying out a polyphasic approach combining morphological aspects and molecular tools is essential for the correct identification of these species. As also shown by Paes et al. (2022), in the present work the analyses of the D1/D2 domain of large subunit rRNA gene revealed that it provided a good resolution to discriminate the Geotrichum species, being considered a good marker for identification of species of this fungus.

The methodology used to carry out the pathogenicity test highlights by Holmes e Clark (2002) suggest the importance of an environment with low oxygenation and high humidity for the reproduction of some of symptoms caused by G. candidum in sweet potato in the field, having knowledge that the souring of sweet potato is not an isolated factor, but the result of a complex of factors like the pathogenic effects of G. candidum in the environment (Holmes & Clark 2002).

The specie Geotrichum candidum was recently reported in Brazil causing sour rot in Melon (Cucumis melo) (Halfeld-Vieira et al. 2020) and Tomato (Solanum lycopersicum) (Paes et al. 2022). In Brazil, sour rot is an important post-harvest disease on fruits and vegetables, the identification of these fungi is the basic strategy for the prevention and control of the disease. To our knowledge, this is the first report of G. candidum causing post-harvest sweet potato sour rot in Brazil.