First report of the association of Genus Chaetomium with roots of Cucurbita maxima

Chaetomium sp. is a fungal genus in the Ascomycota that has a world-wide distribution throughout tropic and temperate regions. Species of Chaetomium are often associated with and classified as both seed-borne and soilborne fungi, which inhabit a variety of terrestrial ecosystems including; agricultural, forests, grasslands, wetlands, and coastal zones. Chaetomium sp. plays an important ecological role as a saprophyte, but also shows strong antimicrobial activities which allow its use as a biological control agent (BCA). Chaetomium sp. has been recorded on the seeds and within the rhizosphere of a variety of genera of the family Cucurbitaceae including; Citrullus, Cucumis, Cucurbita, Lagenaria, and Luffa. Within the genus Cucurbita, Chaetomium sp. has been documented in association with Cucurbita pepo and Cucurbita moschata, however up till now there is no record of association of Chaetomium sp. with Cucurbita maxima. Sampling of Cucurbita maxima roots in the eastern United States resulted in the discovery of a novel relationship between Cucurbita maxima and Chaetomium sp. This manuscript presents the first report of existence of a relationship between Chaetomium sp. and Cucurbita maxima, with the aid of light microscope to image the perithecia and spores produced by this fungus.


Introduction
Chaetomium (Kunze) is a genus of fungi in the Ascomycota that inhabits terrestrial ecosystems, and has a world-wide distribution throughout tropic and temperate regions. Mahdy et al., (1987); Selvi and Balagengatharathilagam, 2014;Gashgari et al., (2016) reported that Chaetomium plays an important ecological role as a saprophyte and as a biological control agent. Various Chaetomium species are known to produce the alkaloid Echinulin, which has been shown to be incredibly toxic to mammals, causing damage to the internal organs including the lung and liver (Barros-Filho et al., 2012).
Cucurbita maxima (Duchesne) is a member of the Cucurbitaceae family, and has been used by human for more than 4000 years as a nutritional source, medicinal plant, and as a storage container (Zhang et al., 2006;Hulse, 2018;Hulse and Braselton, 2019). C. maxima seeds and fruit were disseminated to Europe sometime in the 16 th century, and taken by the European explorers to the Indian sub-continent and Southeast-Asia. Moreover, C. maxima is used in traditional medicine, because it is reported to have anti-diabetic, anti-cancer, and anti-inflammatory properties. In addition, it is also used as a food source by a wide variety of world cultures, due to its high fiber content, diverse carbohydrates, β-carotene, vitamins, alkaloids, minerals, fatty acids, flavonoids, and polysaccharides (Saha et al., 2011;Zhou et al., 2014;Hulse and Braselton, 2019;Maurya et al., 2019).
Since the early 1900's, C. maxima has been widely used as a model organism as it has larger phloem cells than most plants of an equivalent size, which makes it an ideal candidate for different studies involving nutrient movement, nutrient acquisition, cell signaling, in addition to other studies that involve the phloem tissues (Ferriol et al., 2004, Hulse, 2018, Hulse and Braselton 2019. It has also been used as a model organism in the inter-and intraspecific grafting studies, because it has a strong affinity for surviving the grafting process, and grafts easily to the other members of the Cucurbitaceae. Ferriol et al., (2004); Hulse and Braselton, (2019) pointed that currently C. maxima remains a model organism for molecular, nutritional studies, and will continue to be used in more studies because of its multi-million-dollar economic impact in the United States of America (USA).
The current research was designed in order to more fully understand the microbial communities associated with C. maxima, and to investigate the presence of a relationship between Chaetomium sp. and C. maxima. Verification of contact between both organisms will create a point of support for researchers to expand on this knowledge, and further explore the ecological role of Chaetomium sp. in C. maxima's rhizosphere. Due to the health concerns associated with the alkaloids produced by Chaetomium sp., it would be vital to understand more about this novel relationship from the public health perspective. This manuscript provides the first report and evidence of the existence of a relationship between Chaetomium sp. and C. maxima.

Field survey
A survey of nine varieties of C. maxima was conducted in the eastern United States, and traditional staining techniques were used to confirm the presence or absence of associations between Chaetomium sp. and the host. In addition, the field survey was conducted in order to provide further exploration of the colonization mechanisms of Chaetomium sp. in C. maxima.

Samples collection
Five root samples of C. maxima were collected from two farms in South Eastern Ohio, USA; one farm was in West Virginia, while the other farm was in Maryland, USA. These farms were selected based upon their different agricultural practices that covered all current cultivation practices including; certified USDA Organic, uncertified organic, unsprayed, and conventional chemical treatments. The five root samples were collected randomly from three plants of each cultivar, and then these roots were used for staining and microscopic analysis. The different cultivars of C. maxima examined in this study include; Banana, Blue Hubbard, Burgess Buttercup, Dills Atlantic Giant, Rouge Vif d'Etampes, Red Kuri, Sweet Meat, Polar Bear, and Turk's Turban.

Root staining
Root staining was an optimized modification of the Brundrett method (Brundrett et al., 1984), originally used for clearing and staining plant roots. Root samples were heated in 10% (w/v) aqueous potassium hydroxide (KOH) solution for 50 min. at 95 o C. Excess KOH was decanted off, and then 5% hydrochloric acid (HCl) (v/v) was added to neutralize the pH. The samples were kept in 5% HCl for 5 min. at 20 o C. After that, 1% (w/v) Trypan blue was added to the Falcon tubes, boiled at 100 o C for 5 min. on a water bath, and then immediately washed with dist. water. Samples were washed once with 50% lactic acid-glycerol (v/v), cut into 1 cm 2 sections and mounted on glass slides with fresh 50% lactic acidglycerol (v/v), finally stored at 4 o C until imaging.

Light microscopy
Root segments of C. maxima cv. Banana, Blue Hubbard, Burgess Buttercup, Dills Atlantic Giant, Rouge Vif d'Etampes, Red Kuri, Sweet Meat, Polar Bear, and Turk's Turban, were examined for distinct morphological features associated with the genus Chaetomium. Two, 1 cm 2 segments of the root samples were mounted on glass slides containing 50% lactic acid and 50% glycerol. These slides were then examined for several features including; perithecial shape, melanized septate setae, and ascus containing fusiform spores, in addition to other features proposed by Hanlin, (1990); Hanlin (1998). Finally, the samples were imaged using an Olympus AX -70 light microscope.

Results
Cucurbita maxima roots were collected from two farms in south eastern Ohio, USA. Different varieties of C. maxima were sampled from each farm ( Table 1).
Results of investigating the samples of C. maxima demonstrated the presence of Chaetomium sp. perithecia, establishing the existence a relationship between Chaetomium sp. and C. maxima cv. Rouge Vif D' Etampes, in samples collected from the eastern panhandle at Kensho Farms, West Virginia, USA (Fig.  1A-D). As stated before, Kensho farm is an uncertified organic farm that doesn't use synthetic chemicals to treat its plants.

Discussion
Members of the Cucurbitaceae are grown around the world as materials to build musical instruments, water storage devices, as well as for food and nutritional resources. Moreover, in some locations in the world, cucurbits are used for their medical properties (Hulse, 2018;Hulse and Braselton, 2019). The species of C. maxima has been used as a model organism during the current research project, due to its long-term historical use within the agricultural research community (Hulse and Braselton, 2019). Microbial communities associated with C. maxima have not fully been documented, and this manuscript helps elucidate a critical component of the ecosystem that has been missing in the previous literatures (Hulse, 2018;Hulse and Braselton, 2019). Cucurbita maxima is an economically important species and its microbial communities should be studied in more details, because they can be beneficial not only to the plants such as in mycorrhizas, but also to find biological control agents (BCAs) against the potential pathogens (Hulse and Braselton, 2019). This plant belongs to a large family that contains about 800 species, and many of them are of significant economic importance to the farmers of Africa, Asia, Europe, as well as in North and South America.
Chaetomium sp. is an economically important Ascomycete fungus that has a world-wide distribution Novel Research in Microbiology Journal, 2020  in temperate regions. It has been shown to produce toxins that are fatal to mammals, and have antimicrobial properties. Previous studies of Suryanarayanan et al., (2000);Avinash et al., (2013); Rahim et al., (2013) showed the existence of a relationship between Chaetomium sp. and other members of the Cucurbitaceae including; C. pepo and C. moschata, but never recorded in association with C. maxima. This indicates that the Chaetomium sp. reported in this study might not be described before in other studies. Future phylogenic studies using multiple molecular markers should be used to correctly identify the species of Chaetomium discovered during this study. The morphological characteristics alone cannot define a species with high certainty, due to complications in distinguishing between convergent evolution and true monophyletic clades. Hulse and Braselton, (2019) reported that C. maxima has extreme phenotypic plasticity, which accounts for the hundreds or thousands of varieties of this species cultivated throughout the world. Since a relationship has been recorded between Chaetomium sp. and C. maxima cv. Rouge Vif D' Etampes, future studies should be conducted to determine what other varieties of C. maxima are susceptible to colonization. With the important ecological and economic interests in Chaetomium sp., any information that can be collected on this novel relationship is beneficial to the agriculturalists.
It would also be advantageous to determine which other members of the Cucurbitaceae can be colonized by Chaetomium sp., in order to identify the hosts and the alternate hosts of this potential seed borne contaminate. Since Cucurbits are often intercropped, it is important not to cross contaminate seeds stock of Cucurbits that may be susceptible to infection by Chaetomium spp.
Since several members of the genus Chaetomium are well known to produce toxic alkaloids, it is important to identify if the Chaetomium sp. recorded in this study (presented in Fig. 1A-D) is a source of this alkaloid, and screen seeds that will be directly used for human consumption.
Growing C. maxima in the United States has a yearly economic impact of hundreds of millions of Dollars, and any information regarding its associations with economically important microbes should be documented. This study provides the first report and images documenting the existence of an association between C. maxima and Chaetomium sp., and it will hopefully led researchers to better understand the microbial communities associated with the C. maxima agricultural ecosystems.

Conclusion
This study records the first report of Chaetomium sp. in association with the roots of C. maxima. Bright field light micrographs provided the evidence of existence of this relationship in the rhizosphere of C. maxima cv. Rouge Vif D' Etampes grown under uncertified organic conditions in the eastern panhandle of West Virginia, USA. Establishment of a relationship between Chaetomium sp. and the roots of C. maxima can provide basis for future research concerning the ecological role of Chaetomium sp. within the rhizosphere. The Chaetomium sp. discovered in this study should be identified using recent molecular techniques that utilize multiple markers, in order to accurately characterize this isolate. Future research should explore other varieties of C. maxima in order to determine if they harbor a relationship with Chaetomium sp. within their agricultural ecosystems.