1. Introduction
Phaseolus vulgaris L., known as common bean, is the second most important legume in the world for food purposes thanks to its high nutritional value in terms of content of protein, vitamins, zinc, iron, and fiber [
1,
2,
3]. It is widespread over a large area between 52° N and 32° S latitude, at low altitudes until sea level (USA and Europe) and high altitude (South America) [
4,
5,
6]. In Italy, the cultivated area is 22,501 ha (17,161 and 5340 ha for green and dry beans, respectively); the Basilicata Region (South Italy) is invested with 125 ha of dry beans [
7]. “Fagioli di Sarconi” common beans include about 21 different ecotypes protected by the European Union (Commission Regulation (EC) No 1263/96) with the mark PGI (Protected Geographical Indication). These legumes are used as a prevalent healthy food in Italian cuisine, and are cultivated in the National Park of the Agri Valley in Basilicata for dry seed production [
8,
9,
10].
The “Consorzio di Tutela dei Fagioli di Sarconi” has preserved the germplasm on farm and its biochemical and nutraceutical traits have been characterized [
5,
8,
9,
10,
11,
12,
13,
14,
15,
16]; however, research on the response to diseases of common bean ecotypes is limited [
17].
Seed-borne pathogenic fungi and bacteria can inhibit germination, infect the seedlings to death, or reduce plant growth by damaging the roots and vascular system, and affecting the transport of water and nutrients [
17,
18,
19,
20].
The main seed-borne pathogenic fungi that cause losses of yield and quality of common bean are
Alternaria spp.
Aspergillus spp.,
Penicillium spp.,
Fusarium spp.,
Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cav,
Botrytis spp.,
Rhizopus spp.,
Cladosporium spp.,
Rhizoctonia solani (Cooke) Wint [
19,
20,
21,
22,
23,
24]. All these fungi are seed-transmitted and can be preserved in the form of conidia in the coat or as mycelia on the seed surface [
25], at extra- or intra-embryal level, or associated with the seeds as contaminants [
26]. For this reason, all these fungi represent a limiting factor for common bean production in many countries.
According to the classic disease triangle, the host–pathogen–environment interactions are the cause of plant disease occurrence and severity [
27]. Therefore, the environment has been always considered an important ring in plant pathology studies. Currently, climate change is strongly associated with seed-borne fungally induced disease incidence and severity. In fact, climate changes influence the growth, reproduction, survival, spread, pathogenicity, as well as the ability to modify a typical soil microflora, enabling the emergence of new species [
28,
29].
In this context, this study aimed to determine the influence of different cultivation areas on the presence of seed-borne pathogens isolated from seeds of two ecotypes of “Fagioli di Sarconi” common beans, “Ciuoto” and “Cannellino rosso”, collected from two different cultivation areas of the Basilicata Region during the years 2018 and 2019, for the identification of genotypes resistant or little susceptible to seed-borne pathogens and well adapted to environmental changes.
3. Results
3.1. Meteorological Parameters
The rain, temperature and relative humidity data for the Agri Valley territory during the cultivation period, from June to November for both years 2018 and 2019, are summarized in
Table 2 and in
Figure 2.
During the 2019 cultivation cycle the average daily rainfall, equal to 1.69 mm day−1, was lower than the previous year (2.09 mm day−1), but was not significantly different. Regarding the temperatures, only the maximum showed a significantly different value between the two years. In the same way, the minimum and average relative humidity were significantly different between the two years, with values in 2019 being lower than in the previous year. As a consequence, the average evapotranspiration during 2019 was significantly higher with respect to 2018.
3.2. Isolation and Identification of Seed Mycoflora by Washing Test
Eighteen seed-borne fungal species were found contaminating or infecting the two ecotypes of “Fagioli di Sarconi” PGI common bean seeds in the two different areas and during the two years 2018 and 2019 (
Table 3).
Microscopic morphological structures identifying the fungi
Rhizoctonia solani isolated from “Cannellino rosso” ecotype in Paterno (2018),
Colletotrichum lindemuthianum and
Fusarium oxysporum isolated from “Ciuoto” in 2019 in Paterno and Sarconi, respectively, are shown in
Figure 3. In particular,
Figure 3a shows the typical durable moniloid cells of
R. solani; the conidia of
C. lindemuthianum showed in
Figure 3b are typically cylindrical, fusiform or sickle−shaped, hyaline with smooth wall and with two guttules; the macroconidia of
F. oxysporum were individuated by their typical three to five septa, the apical and basal cells being moderately curved and foot shaped, respectively, while the microconidia for their drop shape were without septa (
Figure 3c).
3.3. Identification and Incidence of R. solani on Treated and Untreated Seeds with Blotter Test, and of C. lindemuthianum, F. oxysporum and Bacterial Diseases Causal Agents with between Paper Test
The blotter test was able to identify, in the 1% sodium hypochlorite treated seeds, the following seed−borne fungi: R. solani (both years, ecotypes and localities); Aspergillus niger (2018, Paterno, both ecotypes; 2019, Sarconi, “Cannellino rosso”); F. solani (2019, Paterno, both ecotypes); Penicillium expansum (2018, Paterno, “Ciuoto”).
On the other hand, the following fungi were detected in untreated seeds: Aspergillus flavus (2019, Sarconi, “Cannellino rosso”); A. niger (2018, Paterno, both ecotypes; 2019, Sarconi, both ecotypes); Cladosporium cladosporioides (2019, Sarconi, “Cannellino rosso”); F. solani (2018, Paterno, “Ciuoto”; 2019, Paterno, “Cannellino rosso”); Mucor hiemalis (2018, Sarconi, “Ciuoto” and Paterno, both ecotypes; 2019, Paterno, both ecotypes); Penicillium spp. (2018, Sarconi, “Cannellino rosso”); P. expansum (2018, Paterno, “Ciuoto”); Rhizophus nigricans (2018, Sarconi, “Ciuoto”).
With the between paper test, although specific for
C.
lindemuthianum, the fungus
F.
oxysporum and two bacterial diseases causal agents were isolated and detected, as reported in
Table 4.
The seed-borne pathogenic fungi and bacteria considered the most dangerous in determining losses of yield and quality in the common bean were deeply considered in terms of their disease. The percentage of rhizoctoniosis from
R.
solani, as well as the presence of typical symptoms on integument and cotyledons induced by
C.
lindemuthianum,
F.
oxysporum and bacterial diseases causal agents are depicted (
Table 4).
R. solani was detected in both ecotypes and years. The incidence of rhizoctoniosis in common bean seeds treated with hypochlorite for both years was higher for “Ciuoto” with respect to the “Cannellino rosso” ecotype in both Paterno and Sarconi. On the other hand, during the year 2019, the rhizoctoniosis incidence for all treated seeds decreased.
Conversely, R. solani induced a higher disease incidence in the untreated seeds, in both ecotypes and years of cultivation, in the Paterno area.
As showed in
Table 4, χ
2 test indicated differences between ecotype, year and cultivation area for the incidence of fusarium disease as highly significant. The highest incidence of phytopathy was recorded in 2019 for “Ciuoto” in the Paterno area. In Sarconi, in the same year, the presence of fusariosis on seed was not found.
The incidence of anthracnose symptoms from C. lindemuthianum was evaluated on integument and cotyledons by between paper test. The differences between ecotype, year and area were highly significant, as shown by χ2 test. In particular, for this pathogen, the Paterno area was the most infected one, with the year 2019 being more serious than the previous year and “Cannellino rosso” showing less susceptibility to anthracnose than the “Ciuoto” ecotype.
In summary, the treatment of the seeds with 1% Na−hypochlorite allowed a reduction of most fungal microflora adhering to the seed, such as Aspergillus flavus, but did not eliminate the systemic pathogens infecting the internal structures of the seed, such as C. lindemuthianum, F. solani, and R. solani.
Furthermore, the different environmental factors in the two years and cultivation areas determined a different incidence of the considered diseases
3.4. Macroscopic and Microscopic Characterization of R. solani, C. lindemuthianum and F. oxysporum
The typical damping−off symptoms of
R. solani on the stem of the “Cannellino rosso” common bean ecotype, located in Sarconi (2019) and “Ciuoto”, and located in Paterno (2018), as revealed by blotter test, are shown in
Figure 4a,b.
Figure 4c clearly shows binucleated
R. solani isolates with considerable morphological variability from white to creamy−yellow, with a felty appearance for the white mycelium, while a daily growth rings with variable tonality in the other. The conidiophores branches of the
R. solani isolates resulted fold in the direction of growth and slightly narrowed at the point of insertion on main hypha; moreover, the secondary conidiophore branches reached right−acute angles on the main hypha at maturity (
Figure 4d).
Figure 5a reports typical anthracnose symptoms on seeds detected by between paper test as spots, more or less wide, and surrounded by a purple halo, as well as some macroscopic and microscopic morphological structures identified as the fungus
C.
lindemuthianum isolated from “Ciuoto” ecotype, located in Paterno (2018).
C. lindemuthianum is a mitosporic fungus with four physiological breeds known to have different degrees of pathogenicity compared to common bean cultivars. Colonies are from salmon−gray to mouse−gray in color, as shown in our isolates (
Figure 5c), and produce acervules consisting of a compact layer of cylindrical, hyaline, curved, pigmented phialides (
Figure 5b). The typical acervules of
C. lindemuthianum with dark, scattered, 5–9 septate and pigmented bristles are depicted in
Figure 5d.
The morphological characteristics of
F. oxysporum are reported in
Figure 6. Typical rot seeds were revealed, characterized by the presence of abundant aerial, white, felty, flaky with a purplish central mass mycelium, as also confirmed by the macroscopic appearance of the colonies on the plate (
Figure 6a,b). The macroconidia were slightly curved, with three to five septa, and hooked basal and short apical cells, as is typical of this fungus; even the microconidia appeared to be typically formed at the end of the phialidia, and were abundant, fusiform, reniform, and produced by branched polyphialides (
Figure 6c).
4. Discussion
Seed health is a critical factor in achieving a good product and high yields. The presence of pathogens in or on the seed influences all the plant vegetative and productive phases. Seeds intended for food may not only have little nutritional value, they may also be contaminated with mycotoxins. Therefore, the health of the seed is an indispensable requirement for the productivity and quality of the crop. For the isolation and identification of the seed-borne pathogens of common beans, diagnostic methods with different sensitivities were used. The washing test method allows to identify the spores of the fungi contaminating the seed surface [
32,
33]. Therefore, it proved to be the most effective and sensitive compared to the other ones for this purpose. In fact, the washing test made it possible to detect most of the fungi contaminating the integument and infecting the cotyledons and the embryo. There were 18 seed-borne fungal agents. Most of these (
R. solani,
C. lindemuthianum,
F. oxysporum,
F. solani,
U. appendiculatus) are of great importance due to the damages they cause to the seed in pre− and post−emergence, thus causing economic losses [
46]; the other fungal pathogens detected are no less dangerous (
Alternaria alternata,
Penicillium spp.,
A. flavus,
A. niger,
M.
hiemalis,
Fusarium spp.), and determine substantial problems linked to mycotoxin production during post−harvest storage [
16]. This method also made it possible to identify beneficial fungi (
T.
harzianum and
T. viridae) known to be used as antagonists of harmful pathogens [
47,
48,
49].
The blotter method, unlike the washing test, is more selective towards some pathogens with saprophytic behavior (Aspergillus spp., Cladosporium spp., Botritys spp., Mucor spp., Rhizophus spp.). The treatment of the seeds with 1% Na−hypochlorite allowed a reduction of most of fungal microflora adhering to the seed, but did not make it possible to eliminate the systemic pathogens infecting the internal structures of the seed, such as C. lindemuthianum, F. solani, and R. solani. Nevertheless, the treatment was able to decrease the presence of some conidia and/or mycelia in the coat and on the seed surface, thus decreasing the disease incidence of R. solani. The between paper method, applied as international protocol specific for detecting C. lindemuthianum, revealed its presence in both ecotypes, years and areas.
Regarding thermo−hygrometric data, collected during the entire crop cycle, the average air temperature was 17.20 °C in 2018 and 18.08 °C in 2019, without significant difference between the two years, while the maximum temperatures, equal to 26.13 °C and 27.78 °C during 2018 and 2019, respectively, were significatively different. This finding could explain the prevalent presence of the mesophilic fungal species detected during the year 2018, when their spores were able to germinate at humidity values of 55–75%, such as
Alternaria spp.,
A. alternata,
C. cladosporioides,
Penicillium spp.,
Fusarium spp.,
Colletotrichum spp., and
R. solani [
38,
42].
Temperature plays an important role in sporulation, germination of conidia and in the development of fungal pathogens.
Table 5, built taking into account scientific studies investigating the development of the same isolated and identified phytopathogenic fungi in the current study, clearly indicates that they can develop between −5 and 50 °C, with optimal growth around 25 °C.
In particular, fungal species found on the two “Fagioli di Sarconi” PGI common bean ecotypes seeds, belonging to thermophilic fungi (development at maximum temperature of 45–50 °C, and minimum growth temperature not lower than 20 °C), were A. niger and M. hiemalis. The detected mesophilic fungi (development between 10 and 40 °C, with optimum at 25 °C) were Alternaria spp., A. alternata, Aspergillus spp., A. flavus, C. lindemuthianum, F. oxysporum, F. solani, R. solani, U. appendiculatus and Trichoderma spp. Finally, C. cladosporioides, Botrytis spp., R. nigricans, Penicillium spp., P. expansum were the detected cryophilic fungi (optimum development between 5 and 10 °C).
For the years 2018 and 2019 the mean minimum relative humidity was 42.39% and 37.58%, respectively, while the average relative humidity values were 75.09% and 71.25%. These values indicate a small, but significant difference between the two years. The humidity, as well as air temperature, is important for the development of phytopathogenic fungi. In particular, xerophilic fungi prefer dry environment, where spores germinate with low relative humidity values (<55%), as indicated in
Table 6, built in the same way of that of temperature.
Aspergillus spp.,
A. niger and
A. flavus were the xerophilic fungi detected in the current study.
We found that most seed-borne isolated fungi were mesophilic with optimal growth relative humidity values between 55 and 75%, as mentioned above, including Alternaria spp., A. alternata, C. cladosporioides, Penicillium spp., Fusarium spp., Colletotrichum spp. Among the hygrophilous fungi, having spores that germinate with humidity above 75%, we found R. solani, Botrytis spp., and Mucor spp.
R. solani is a polyphagous pathogen that attacks different plant portions from the soil surface. The early symptoms are detected on the cotyledon leaves, at the base of the stem with typical dropsy areas followed by bottlenecks and brown spots on the young roots. This pathogen multiplies by repeatedly spreading its mycelium. In fact, the infections occur due to the mycelium, which is able to spread into the young tissues of the plant. The preservation of the fungus occurs in the soil as mycelium and as pseudosclerotia able to survive 6–7 years.
R. solani has a wide distribution in temperate regions, where the optimal development temperature ranges from 15 to 22 °C, with frequent rainfall [
53].
The different temperature and relative humidity in the two reference years explains the different disease incidence of some seed-borne fungi. In fact, during 2018, characterized by lower maximum temperature and higher minimum and average humidity than to 2019, we recorded a greater rhizoctoniosis incidence induced by R. solani for both ecotypes and areas.
Anthracnose is the most severe fungal adversity of common bean. The etiological agent
C.
lindemuthianum is kept in the seed and in the soil as mycelium for up to 5 years [
19].
F. oxysporum is a pathogen perpetuated in the soil, where it is kept for several years thanks to the chlamydospores [
24]. The development of these two pathogens, mesophilic species, is affected by high rainfall and humidity. For this reason, it is possible also for anthracnose and fusariosis to explain the higher incidence of the disease in the year 2019 compared to the previous one as being due to the lower humidity.
In addition, concerning the incidence of anthracnose by
C. lindemuthianum, of fusariosis by
F. oxysporum and of bacteriosis, the results highlighted that the “Cannellino rosso” ecotype was less susceptible to plant diseases compared to the “Ciuoto” one. The reasons are to be found not only in the higher integumental thickness of “Cannellino rosso” with respect to others local ecotypes [
12,
54], but probably also in the higher content of polyphenolic compounds (hydrolysable tannins and condensed tannins) present in the integument, which are considered protective factors for the seed against seed-borne pathogens [
55,
56,
57] and also considered potentially able to constitute a defense mechanism against fungal attack and mycotoxin production in beans [
58]. Conversely, only the Na−hypochlorite treatment induced less susceptibility to rhizoctoniosis by
R. solani in the “Cannellino rosso” ecotype.