DIVERSITY AND BIOTIC ACTIVITY OF FUNGI COLONIZING PUMPKIN PLANTS (CUCURBITA PEPO L.) GROWN IN THE FIELD

Agnieszka Jamiołkowska¹, Ali H. Thanoon^2
1 Department of Plant Protection and Quarantine, Faculty of Horticulture and Landscape Architecture, University of Life Sciences in Lublin, Poland
² Department of Plant Protection, Faculty of Agriculture and Forestry University of Mosul, Mosul, Iraq

ABSTRACT

Pumpkin (Cucurbita pepo L.) is one of the popular seasonal vegetables cultivated in the world under different climatic conditions. This crop is important as a vegetable and medicinal plant used as a component in the diet. The aim of the experiment was to estimate the biodiversity of fungi colonizing pumpkin plants cultivated in the field and determine the strength of the interaction in communities of fungi, by using the biotic activity test. The experiment was set up in the organic farm in Zezulin (Lublin province, Poland) where the pumpkin plants ‘Bambino’ were cultivated in 2010 and 2012. The leaves, stems and roots of pumpkin were collected for mycological analysis at the beginning of fructification (71 BBCH). Laboratory test showed that predominating fungi colonizing plants were Alternaria alternata, Fusarium culmorum, Gibberella intricans, F. oxysporum and Tanatephorus cucumeris.

The biotic interactions between the most important species fungi such as A. alternata, F. culmorum, F. oxysporum and other fungal species were studied using the biotic series method. Trichoderma spp. were found as the most effective and positive antagonists against Alternaria alternata, F. oxysporum and F. culmorum. It was show that A. alternata and F. oxysporum were weak competitors because their growth was limited by numerous fungi such as Trichoderma hamatum, Tanatephorus cucumeris and other fungi used in the test. F. culmorum was strong competitor because it limited the growth of other fungi from phyllosphere of pumpkin plants, however its growth can be inhibited by a great number of Trichoderma spp. colonies.

Key words: biodiversity of fungi, squash, Alternaria alternata, Fusarium spp., Giberella spp., Trichoderma spp., biotic series method.

INTRODUCTION

Pumpkin (Cucurbita pepo L.) is a species native to North America but cultivated worldwide. The Polish food industry is interested by pumpkin plants production. This vegetable is useful, easy to grow and achieve high yields [29]. This is related to breeding the novel, Polish cultivars which are characterized by higher contents of dry matter, the best thickness of fruit flesh and large content of nutrients [23]. Now, as a result of research conducted by the Department of Genetics, Breeding and Biotechnology of University of Life Sciences in Warsaw, novel cultivars of pumpkin such as ‘Amazonka’, ‘Ambar’, ‘Justynka F1’, ‘Karowita’, and ‘Otylia F1’ are cultivated [22].

Pumpkin is important as a vegetable and medicinal plant used as a component in the diet [23]. Seeds are found to have some benefits for diabetes, antimicrobial activities and anticancer properties. The nutrient profile of pumpkin seeds showed that they are low in calories, and the seeds are rich source of vitamin A, B1, B2, B3, B6, B12, C, D, E, K, pantothenic acid, minerals (Ca, Mg, F, P, Fe, Se, Zn), carotenoids (lutein, zeaxanthin and β-carotene), amino acids and many other nutrients. They also contain wide variety of antioxidants phytonutrient [3, 7, 31]. Carotenoids are natural pigments suitable yellow, orange or red colour of food products. Most of them have health-promoting action. An example is lutein, which is important habits due to the antioxidant and anticancer activity and protect the body against cardiovascular disease – Polish, novel pumpkin cultivars ‘Justynka’, ‘Amazonka’ and ‘Ambar’ are rich in natural carotenoids and might be widely used in food industry as a source of bioactive, health promoting phytochemical compounds [22].

Pumpkin is cultivated in many countries as a vegetable. In Poland, two pumpkin species are grown and harvested to the phase of physiological maturity: Cucurbita maxima L. and Cucurbita pepo L. [8, 21]. Cucurbit vegetables grown in the field condition is usually infected by pathogens specific to Cucurbitaceae family species, as well as organisms characteristic of other plantations [15, 26]. Several fungi have been identified as Alternaria alternata and A. cucumerina (alternaria leaf spot), Pseudoperonospora cubensis (downy mildew), Podosphaera xanthii (powdery mildew), Colletotrichum orbiculare (anthracnose), Cladosporium cucumerinum (scab or gummosis), Septoria cucurbitacearum (septoria leaf spot), Pythium spp. (damping-off), Rhizoctonia solani (damping-off), and Fusarium spp. (damping-off and wilt) [5, 6, 15, 26, 36]. Characterization of the population structure of fungal communities including pathogens, is important for understanding the biology of the pest and for development of disease-control strategies.

The aim of the study was to estimate the species composition of the fungi colonizing pumpkin plants (Cucurbita pepo L.) grown in the field, with particular emphasis on pathogenic species, and determine the strength of the interaction in communities of fungi, by using the biotic activity test.

MATERIAL AND METHODS

Field experiment
The experiments were conducted in 2010 and 2012 in an horticultural farm situated in Zezulin (51°21′22°50′E) (Lublin province), in Poland. The object of study was the plants of pumpkin (Cucurbita pepo L.) ‘Bambino’ cv. The seeds of pumpkin were sown in the field 2nd decade of May, at the spacing of 150 cm in a row and 150 cm between rows. In the experiment was using the method of random squares in four replications (40 plants in combination). Before setting up the experiment, mineral fertilization was used according to the recommendations for Cucurbitaceae, after a previous analysis of the soil. During the vegetation season the plants were manually weeded and they were not chemically protected.

Mycological plants analysis.
The  mycological analysis of plants was  made  at the beginning  of  fructification (71 BBCH)  in  the  laboratory  of  the  Department of Plant Protection and Quarantine of the Life Sciences University in Lublin. Four plants, randomly selected, from combinations were collected for mycological analysis. Leaves (petiole and lamina), stems and roots of pumpkin were analyzed in laboratory according to the method described by Jamiołkowska [10]. For each experimental treatment 10 Petri dishes with the plant material (10 plant fragments per dish) were prepared and incubated at 20–22°C for 7 days in dark. The obtained colonies of fungi were transferred to potato-dextrose agar (PDA Difco). The grown colonies were determined to the species by means of the available monographs and mycological keys.

Biotic activity test.
The fungi for biotic activity test were selected as a result of mycological analysis of pumpkin plants in 2010 and 2012. Isolates of tested fungi such as Alternaria alternata – LD1 (from leaf), Fusarium culmorum – ŁD15 (from stem), Fusarium oxysporum – KD8 (from root) and single isolates of other species fungi were chosen randomly from the collection obtained from mycological analysis. Test fungi used for biotic test came out from leaves (Epicoccum nigrum LD6, Gibberella avenacea AL3, G. intricans AL2, Mucor hiemalis AL5, Trichoderma hamatum LD4) and stems (Alternaria alternata AŁ2, Aureobasidium pullulans AŁ4, G. avenacea AŁ1, G. intricans DŁ3, T. hamatum DŁ22), as well as from roots (A. alternata KD5, A. pullulans AK17, Chaetomium globosum AK22, G. avenacea DK18, F. culmorum DK2, G. intricans DK15, F. solani AK23, Tanatephorus cucumeris AK5, T. hamatum KD6, T. harzianum DK13). The maximum number of fungal species was taken for this study, irrespective of frequency of their isolations from plants in the study period. The interaction of the fungi was studied by using the biotic series method on PDA (Difco) medium [19]. Two discs of 3 mm in diameter from 14-day-old cultures, one of tested fungi (A. alternata, F. culmorum and F. oxysporum) and one of the fungus representing the studied community (test fungi), were taken. They were placed mycelium down, 2 cm apart in the central of the Petri dish, on the solidified medium. The dishes with single fungi species constituted the control. For each experimental combination, 4 dishes were considered which were treated as replications. They were kept in a thermostat at 24°C in the dark. The biotic effect was estimated on the basis of an 8-degree scale after 10 days of common growth. While evaluating the biotic effect, the overgrowth of the fungus colony by the accompanying fungus, the occurrence of the inhibition zone between two colonies and growth inhibition of the colony of one of the fungi were taken into consideration [18, 19].

The growth of tested fungi (isolates A. alternata, F. culmorum, F. oxysporum) of the accompanying fungi representing the studied communities was expressed as an Individual Biotic Effect (IBE) [19]. Next, the General Biotic Effect (GBE) was estimated which was the product of the individual biotic effect and the multiplicity of the occurrence of particular fungi species. The algebraic sum of general biotic effects made it possible to determine the Summary Biotic Effect (SBE). A positive value of IBE indicates the growth inhibition of the pathogen, whereas a negative value of IBE points to the lack of growth inhibition of the pathogen’s colony. “0” value means a neutral effect of both fungi on each other [19].

RESULTS AND DISCUSSION

Meteorological conditions during 2-year experiment were presented in Figure 1. Vegetation season 2010 was warm and was characterized by high amount of precipitation than 2012, except in September 2010, when the temperatures were relatively low. High temperatures were also recorded during the growing season of 2012, when average monthly temperatures were higher than long-term average. This season can be described as dry, and the rainfall of this year was below the long-term average, except in October (Fig. 1).

Biodiversity of fungi on pumpkin plants
As the result of mycological analysis 407 isolates of fungi represented by 13 species were obtained. From the aboveground parts of plants (leaves and stems) isolated 345 colonies from 8 species, among which dominated: Alternaria alternata, Fusarium culmorum and Gibberella intricans (Tab. 1–2, Fig. 2). In total, 162 fungal isolates from 11 species were isolated from roots pumpkin among which dominated F. oxysporum and Tanatephorus cucumeris (Tab. 3). Greater species diversity was observed within population in 2010 than 2012. Saprotrophic fungi like Aureobasidium pullulans, Epicoccum nigrum, Trichoderma hamatum, T. harzianumand Mucor hiemalis were also obtained (Tab. 1–3).

A. alternata seems to be the main fungus that colonized the leaves and stems of pumpkin (Fig. 2). It constituted in total 46.1% population of fungi on the leaves and 26.1% populations on the stems (Tab. 1–2). The species was isolated more abundantly in 2012 than in 2010 (Tab. 1). The fungus is dangerous for plants, because it causes strong leaf necroses and dramatic changes in the plant photosystem [2, 20]. It is a polyphagous species which in worse condition of the plants causes irregular, necrotic spots covering a considerable part of the plant [12, 13, 32]. This species – called a “pathogen weakness” – can colonize the plants’ aboveground parts without any symptoms. During the infection process it produces toxins that have a destructive effect on the host plant [27, 28]. Development of A. alternata on plants is determined by weather conditions [28]. Warmer and drier vegetation season in 2012 favored the fungus development on aboveground parts of plants.

Fusarium culmorum and Gibberella intricans  were another species that occurred in big numbers on the aboveground part of pumpkin (Tab. 1–2, Fig. 2). Fusarium spp. are important in pathogenesis of a number of cultivated plants [12, 14, 16]. F. culmorum was abundantly isolated from stems of pumpkin in 2012 and making up 40.6% the total fungi obtained from this part of plants (Tab. 2). Several authors [15, 17] reported strong pathogenic properties of F. culmorum towards many vegetables and spice plants (zucchini, pepper, lemon balm, common thyme) and cause dumping off. F. culmorum is known for producing deoxynivalenol which is not only responsible for the development of tissues necrosis but is also harmful to human beings [9]. G. intricans  was another species that occurred in big numbers on the aboveground part of pumpkin. It was isolated in large numbers of colonies and constituted 22.3% of total population on the leaves and 24.8% of total population on the stems (Tab. 1–2). Jamiołkowska and co-authors [15] reported on strong pathogenic properties of F. equiseti towards zucchini seedlings. Harmfulness of F. equiseti manifests with the inhibition of seedling growth and root necrosis. F. equiseti produces equisetin, substances having antibiotic activity against certain Gram – positive bacteria and phytotoxic activity to seeds and seedlings of certain plants which may disrupt the transport of ATP by mitochondrial membrane [35]. Abundant population of G. intricans and F. culmorum on pumpkin needs to be verified for its pathogenicity to that plant.

Among fungi isolated from pumpkin roots Fusarium genus is worth mentioning (Fig. 2). Most frequently isolated species was F. oxysporum (Tab. 3). The species was isolated more abundantly in 2012 rather than in 2010 (Tab. 3). F. oxysporum induced Fusarium wilt in C. pepo has been reported in Mexico, Iran and Greece [5]. Infestation by F. oxysporum leads to plant’s wilting of the foliage and withering of older leaves, and dying of plants. Choi and co-authors [6] describe this species as main pathogens of zucchini plants in Korea. The harmfulness of F. oxysporum towards zucchini seedlings was confirmed by pathogenicity test [15]. Necrosis of seedling roots and stunting of plants is due to colonizing the underground organs and xylem [25].

Tanatephorus cucumeris was another species that occurred in big numbers on the roots (47 colonies, in 2010) and accounted for 29.0% of the population of fungi isolated from underground parts of plants (Tab. 3). Rhizoctonia solani is considered to be one of the factors causing the rot of seedlings of cultivated plants [16]. Jamiołkowska and Sawicki [13] show that R. solani is a fungus most frequently isolated from zucchini plants cultivated in tunnel plastic conditions than in the open field.

Apart from the potentially pathogenic species, the saprotrophic fungi occurred in the community of fungi colonizing pumpkin plants. Trichoderma hamatum, T. harzianum and Aureobasidium pullulans were isolated from underground parts of pumpkin (Tab. 3). According to many authors [1, 30] presence of these fungi in a habitat is always a positive fact. A. pullulans is a desirable species in the community, because as reported Rotem [28], it is a strong antagonist and primary colonizer of many plants. It is also used as a biological control agent. It is a widely spread epiphyte occurring in the phyllosphere of many plant species. It is commonly known as “black yeast”. The fungus is connected with different environments, both air and water ones. Certain isolates of A. pullulans show antagonistic activity towards phytopathogenic fungi such as Botrytis cinerea, Penicillium expansum and Pezicula malicorticis and it is used in the protection against apple diseases [33]. Basing on the strain A. pullulans L47 obtained in the southern part of Italy from the surface of grapevine fruits, a biopreparation with the commercial name of Boni Protrect Forte GmbH. It contains A. pullulans blastospores stored in the natural medium [33].

Effect of biotic test
Among components of communities isolated from leaves, all species reduced the A. alternata growth. Their Summary Biotic Effects (SBE) were positive in the both studied years (+ 304 in 2010, +318 in 2012) (Tab. 4). The highest inhibitory effect on the growth of A. alternata was caused by T. hamatum and M. hiemalis, because their IBE reached +7 (Tab. 4). We also observed that G. avenacea and G. intricans limited the growth of studied isolate and their IBE’s were respectively +5 and +4 (Tab. 4, Phot. 1). Fungal communities colonizing leaves was able to reduce  A. alternata growth. The positive IBE values obtained in the present study indicate that A. alternata is a weak competitor and its growth is effectively inhibited by other fungi. This may suggest that presence of A. alternata on surface of pumpkin leaves is not dangerous when the plant is in good condition. 

Fungi isolated from pumpkin stems in 2010 were not able to reduce F. culmorum growth (SEB -256), whereas in 2012 the growth of test fungi inhibited growth of this pathogen (SEB +23) (Tab. 5). The numerous negative IBE obtained in biotic study test indicated that F. culmorum can be strong competitor and may inhibit the growth fungal species colonizing pumpkin stems such as A. alternata, A. pullulans and  G. avenacea (Tab. 5). This may also suggest that during simultaneous inhabitation of pumpkin organs by F. culmorum can be also a cause of disease. It was confirmed by pathogenicity tests with F. culmorum isolates and zucchini seedlings [15]. The positive SEB value of tested fungi towards F. culmorum in 2012 shows a strong inhibiting effect of fungi from genus Trichoderma. It was due with presence of many isolates of T. hamatum in 2012 which IEB was +7 (Tab. 5). The colonies of the studied Trichoderma sp. completely overgrew the inoculums of tested fungi and made their growth and sporulation impossible. This phenomenon have a positive and practical aspect in biological control against plant pathogens. Strong competitive abilities of Trichoderma spp. resulting from the production of endo- and exoenzymes, toxic metabolites and from overparasitism [1, 4, 24, 30].

Among fungi achieved from roots: G. avenacea, T. cucumeris and Trichoderma spp. strongly reduced F. oxysporum growth. The Individual Biotic Effects (IBE) above mentioned species towards pathogens were +7 (Tab. 6). Other fungi from the communities did not reduce the pathogen growth showing neutral (F. solani) or negative IBE (G. intricans, A. alternata, A. pullulans) (Phot. 2). Communities originating from roots could inhibit F. oxysporum growth, because their SBE’s were positive (Tab. 6). Trichoderma spp. was the most effective antagonist of Septoria carvi [17]. In studies T. cucumeris should be recognized as effective antagonist of F. oxysporum on the basis of the determined IBE values. However in the aspects of plant protection this fact should be estimated negatively, since the mentioned fungal species are pathogen that are dangerous to Cucurbitaceae plants [15]. T. cucumeris developed their mycelium intensively and formed sclerotia on the whole surface of the F. oxysporum colony and on the culture medium.

The present study showed that SBE values of tested fungi were positive towards A. alternata and F. oxysporum. This suggests that theirs growth can be inhibited in the phyllosphere of pumpkin. It is worth noticing that positive values were obtained in the case of F. oxysporum, which belong to fast growing, toxin-forming fungi pathogenic towards a lot of vegetable plants [11, 15, 34]. Negative IBE value of test fungi towards F. culmorum can indicate strong competitive condition of this pathogen and the risk of disease. However, the presence of many colonies of Trichoderma spp. can limit the pathogen growth. Communities that are rich in high number of antagonistic fungal species, are able to reduce the pathogen growth, therefore it is important – while performing chemical protection – to apply selective preparations that would not destroy antagonistic species. We can suppose that in the Polish climatic condition the community of fungi colonizing the pumpkin plant grown in the field, will inhibit the developing of pathogenic fungi..

CONCLUSION

1. Alternaria alternata, Fusarium culmorum  and Gibberella intricans are the most frequency species fungi colonizing the phyllosphere of pumpkin plants cultivated in the field.
2. The most important species of fungi isolated from pumpkin roots are Fusarium spp. and Gibberella spp., especially F. oxysporum.
3. A. alternata and F. oxysporum, are a weak competitors and theirs growth was effectively inhibited by communities of fungi developing the leaves and the roots.
4. F. culmorum is a strong competitor colonizing the pumpkin stems, however this growth can be inhibited by a great number of Trichoderma spp. colonies.
5. Community of fungi colonizing the pumpkin plant grown in the field, will inhibit the developing of pathogenic fungi of this plant.

REFERENCES

Accepted for print: 19.12.2016

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