Genetic diversity among wild and cultivated germplasm of the perennial pasture grass Phalaris aquatica, using DArTseq SNP marker analysis

Phalaris aquatica L. (phalaris) is a cool-season perennial grass originating from the Mediterranean Basin, north-west Africa and Middle Eastern regions that is used for livestock agriculture mainly in temperate areas with dry summers. It has been the subject of breeding programs in Australia, South America, New Zealand and the USA. Increased knowledge of relationships between wild and cultivated germplasm through use of molecular markers has the potential to facilitate future breeding gains. For this purpose, we conducted an analysis of P. aquatica by using 3905 polymorphic DArTseq SNP markers. Genetic diversity as measured by expected heterozygosity was similar for wild (HE = 0.14; n = 57) and cultivated (HE = 0.13; n = 37) accessions. Diversity in wild germplasm was generally continuous in nature, largely related to geographical location, with a division at the broadest scale into eastern and western clades, with more admixture in the western than the eastern clade. Structure analysis of wild germplasm indicated six subpopulations consistent with country of origin, with some admixture among subpopulations likely resulting from natural and human influences. There were nine subpopulations among wild and cultivated accessions combined. This population structure should be considered if genomic selection is applied in P. aquatica. Analysis of molecular variance indicated that 71% of the genetic variation occurred within subpopulations and 29% among subpopulations. Genetic distances were low among cultivated germplasm from most countries except the USA, which was more distinct. Evaluation of material from the US pool by breeding programs in other countries, and additional material from the less utilised eastern clade, may be worthwhile.

Changes in field concentrations of five phalaris alkaloids and their association with toxicity in pastures of Victoria, Australia

Phalaris aquatica is known to cause toxicity in livestock in the form of acute or chronic staggers or sudden death neurological (SDN) syndrome. Breeding of cultivars that produce lower concentrations of suspected alkaloid toxins has been conducted, but these cultivars continue to cause staggers and SDN toxicity. Field samples of grazed phalaris pasture were collected during one growth season (February–June 2016), and from pastures where cases of staggers and/or SDN had occurred in previous years, and immediately after two cases of toxicity. Pasture collected from a paddock where a case of SDN occurred 4 days prior had elevated levels of 5-methoxy-N,N-dimethyltryptamine (5-MeO DMT) and slightly elevated levels of dimethyltryptamine (DMT) compared with other collections from the region. Pasture collected from a paddock at the time of a case of phalaris staggers did not have elevated levels of the quantified alkaloids. Across the measurement period, potentially toxic alkaloids gramine, hordenine, DMT and 5-MeO DMT were observed to decrease in concentration, whereas β-carboline (norharmane) was not detected in any sample. Excessive drying out of dormant plants was hypothesised to be a risk factor for phalaris toxicity. Continued management of potentially toxic phalaris pasture could include measures to manipulate the physiological processes that result in increased toxic alkaloids, including methods to reduce drying out of dormant phalaris plants, and managing stocking rates and grazing species to mitigate potential toxicity.

Characterisation of Sardinian germplasm of the perennial pasture grass Phalaris aquatica

Germplasm of the perennial pasture grass Phalaris aquatica L., from Sardinia, Italy, is a potentially valuable source for grass breeders owing to climatic similarities to regions where P. aquatica is used, a relatively high incidence of acidic soils, and exposure to prolonged grazing pressure. At field sites in south-eastern Australia, Sardinian accessions were compared as spaced plants and drill-rows with accessions from southern Europe and north-western Africa and with commercial cultivars. They were also evaluated in grazed swards at three sites over 4 years under conditions that challenge persistence, including heavy grazing pressure, acid soils and drought. Morphologically, the accessions were comparatively dense, fine and short, with similarities to southern European accessions and cultivars of the cv. Australian type. However, they were earlier heading and more summer-dormant, particularly those from southern Sardinia. In drill-rows, Sardinian accessions were later heading and less productive in winter than accessions from Morocco. In swards, Sardinian accessions had lower seedling vigour and winter growth potential than modern winter-active cultivars. However, they were dense and persistent under high grazing pressure, and some accessions survived better than all cultivars on an acid, low-fertility soil. Developing cultivars that are superior to the cv. Australian type with acceptable seed production and alkaloid levels presents a challenge to breeders. However Sardinian germplasm offers a range of maturity times combined with higher levels of summer dormancy and a grazing-tolerant morphology, attributes that may expand the area of adaptation of the species into the hotter and more drought-prone margin.

Recovery from short-term complete submergence in temperate pasture grasses

The ability to recover from the impact of short-term submergence was assessed on four widely used grasses in pastures: Dactylis glomerata L., Bromus catharticus Vahl., Schedonorus arundinaceus Schreb. (syn. Festuca arundinacea) and Phalaris aquatica L. Six-week-old plants were subjected to a 5-day complete submergence in clear water, followed by a 15-day recovery period. Dry mass after submergence, shoot and root growth, number of tillers per plant, leaf stomatal conductance and leaf greenness during recovery were assessed. Dactylis glomerata and B. catharticus were sensitive to submergence, showing very low relative growth rate (RGR) of shoots and roots during recovery (37–67% lower than controls) along with early leaf senescence and persistent partial stomatal closure. Schedonorus arundinaceus exhibited an intermediate tolerance, sustaining high RGR of shoots (similar to controls) and fully adjusting its leaf functionality during recovery despite being affected during submergence (40% decrease in dry mass and 37% in tiller number). Phalaris aquatica performed outstandingly, with dry mass unaffected by submergence, and unaltered stomatal conductance, leaf greenness, tillering and shoot growth during recovery. Therefore, in areas where flooding can often cause plant submergence, P. aquatica is recommended whereas the other species are not, because they may be outcompeted by flood-tolerant species.

Αειφορική παραγωγή βιοχημικών προϊόντων από την αξιοποίηση της βιομάζας του είδους Phalaris aquatica L.

Η παγκόσμια κατανάλωση ενέργειας προβλέπεται να αυξηθεί περίπου 35% για την περίοδο 2010-2040 ενώ για την ίδια περίοδο ο παγκόσμιος πληθυσμός θα φτάσει τα 9 δις ανθρώπους. Σήμερα οι αυξημένες ενεργειακές ανάγκες συνεχίζουν να καλύπτονται κυρίως από τα ορυκτά καύσιμα (μη ανανεώσιμες πηγές ενέργειας). Η μείωση των αποθεμάτων των ορυκτών καυσίμων, το αυξημένο κόστος εξόρυξής τους καθώς και οι δυσμενείς επιπτώσεις στο περιβάλλον εξαιτίας των αυξημένων εκπομπών CO2, οδηγούν στην ανεύρεση άλλων πηγών ενέργειας όπως είναι οι ανανεώσιμες. Η λιγνοκυτταρινούχα βιομάζα, η οποία αποτελείται από τρία βασικά δομικά συστατικά, την κυτταρίνη, τις ημικυτταρίνες και τη λιγνίνη, αποτελεί μια υποσχόμενη ανανεώσιμη πηγή για την παραγωγή ενέργειας, βιοκαυσίμων και χημικών ενώσεων υψηλής προστιθέμενης αξίας. Μια από τις βασικές διεργασίες για την αξιοποίηση της λιγνοκυτταρινούχας βιομάζας είναι η βιοχημική οδός, όπου μέσω των σταδίων της προεπεξεργασίας της βιομάζας, της ενζυμικής υδρόλυσης της κυτταρίνης που περιέχει προς γλυκόζη και της αλκοολικής ζύμωσης της παραγόμενης γλυκόζης, οδηγεί στην παραγωγή βιοαιθανόλης.Στην παρούσα διδακτορική διατριβή χρησιμοποιήθηκε η βιομάζα του αγρωστώδες είδους Phalaris aquatica L. προκειμένου να αξιολογηθεί ως ενεργειακή καλλιέργεια ως προς την ικανότητα προσαρμογής του σε περιοχές με διαφορετικές κλιματικές συνθήκες και την παραγωγή βιοενέργειας αλλά και χημικών ενώσεων υψηλής προστιθέμενης αξίας. Για το σκοπό αυτό εγκαταστάθηκε μια νέα καλλιέργεια φαλαρίδας, όπου αξιολογήθηκε η παραγωγή υπέργειας βιομάζας και η συγκέντρωση του λιγνοκυτταρινικού περιεχομένου στο στάδιο της ωριμότητας του φυτού. Έπειτα αξιολογήθηκαν μέθοδοι προεπεξεργασίας της βιομάζας οι οποίες είναι οικονομικές και έχουν μικρό περιβαλλοντικό αντίκτυπο. Οι μέθοδοι αυτοί είναι η υδροθερμική, η υδροθερμική με καταλύτη οξικό οξύ και η βιολογική. Ο στόχος της προεπεξεργασίας είναι η καταστροφή της δομής των μικροϊνιδίων των δομικών πολυσακχαριτών της βιομάζας με την υδρόλυση και διαλυτοποίηση των ημικυτταρινών, ενώ η ενζυμική υδρόλυση που ακολουθεί σακχαροποιεί την κυτταρίνη και οδηγεί στην παραγωγή γλυκόζης. Η ένταση των πειραματικών συνθηκών (θερμοκρασίας και χρόνου) εκφράστηκε με τον συντελεστή δριμύτητας logR0. Τέλος αξιολογήθηκε η θερμογόνος ικανότητα του φυτού.Η υδροθερμική προεπεξεργασία είχε ως βασικό αποτέλεσμα την εκλεκτική απομάκρυνση των ημικυτταρινών, οι οποίες ανακτήθηκαν στα υγρά προϊόντα κυρίως ως μίγμα διαλυτών ολιγομερών/μονομερών ξυλόζης και αραβινόζης προσφέροντας παράλληλα μεγαλύτερη προσβασιμότητα και διαθέσιμη επιφάνεια κυτταρίνης για τη δράση των ενζύμων. Η μέγιστη απομάκρυνση των ημικυτταρινών από τη βιομάζα συνέβη σε μέτριες και υψηλές συνθήκες διεργασίας (logR0 > 4,12), με ποσοστό της τάξης του 90%. Στις υψηλές τιμές logR0 επιτεύχθηκε η πλήρης σχεδόν απομάκρυνση των ημικυτταρινών, όμως το μεγαλύτερο ποσοστό της παραγόμενης ξυλόζης και των υπολοίπων πεντοζών μετατράπηκε σε παραπροϊόντα (φουρφουράλη, HMF, κτλ.) μέσω αντιδράσεων αφυδάτωσης και οξείδωσης. Στις συνθήκες 190οC, 45min (logR0 4,3) παρατηρήθηκε η υψηλότερη τιμή ανάκτησης γλυκόζης με τιμή που έφτασε στα 5,237 mg/ml στις 72 ώρες δράσης των ενζύμων, καθώς και πολύ ψηλή μετατρεψιμότητα της κυτταρίνης σε γλυκόζη (90%). Στην υδροθερμική επεξεργασία με καταλύτη οξικό οξύ οι ημικυτταρίνες ήταν και εδώ το κλάσμα που απομακρύνθηκε εκλεκτικά, αλλά αυτό συνέβη σε ηπιότερες συνθήκες (logR0 > 3,24). Η χρήση του οξέως ευνόησε την αποδιάταξη της λιγνίνης καθώς και τη διάσπαση των ολιγομερών σακχάρων σε μονομερή. Οι συγκεντρώσεις των παραπροϊόντων που σχηματίστηκαν ήταν σε υψηλότερα επίπεδα σε σχέση με τη μη χρήση οξέως. Η συγκέντρωση του οξέως καθώς και το επίπεδο των ενζύμων φάνηκε να μην επηρεάζουν σημαντικά τις αποδόσεις σε γλυκόζη και τη μετατροπή της κυτταρίνης. Ως βέλτιστος συνδυασμός συνθηκών σ’ αυτή τη μέθοδο προτείνεται ο 190οC, 15min με 3% συγκέντρωση οξέος. Στη βιολογική μέθοδο προεπεξεργασίας χρησιμοποιήθηκε ο μύκητας Pleurotus ostreatus σε υπόστρωμα φαλαρίδας, ο οποίος αποδόμησε επιλεκτικά το κλάσμα της λιγνίνης καθώς και τις ημικυτταρίνες (περίπου 50%), κάνοντας ευκολότερη την πρόσβαση των ενζύμων στην κυτταρίνη. Η μεγαλύτερη αποδόμηση των δομικών συστατικών της βιομάζας καθώς και η μεγαλύτερη συγκέντρωση γλυκόζης κατά την ενζυμική υδρόλυση επιτεύχθηκε στο τέλος της καλλιεργητικής περιόδου του μύκητα (120 μέρες). Η μέγιστη μετατρεψιμότητα της κυτταρίνης σε γλυκόζη έφτασε το 88,1%. Τέλος αξιολογήθηκε η θερμογόνος ικανότητα του είδους. Από τα αποτελέσματά μας γίνεται φανερό πως η μέγιστη θερμογόνος δύναμη επιτυγχάνεται σε καλλιέργεια μικρής ηλικίας, λόγω του υψηλού λιγνοκυτταρινικού περιεχομένου, με τιμές που φτάνει τα 19,62 ΜJ/kg ξηρής ουσίας.

Effect of Phalaris aquatica on the abundance and diversity of vertebrate animals in Mediterranean coastal grasslands in California

Harding grass (Phalaris aquatica), an invasive non-native species of bunchgrass, has been introduced to grasslands in many regions of California, particularly those with a history of disturbance, such as tilling and grazing. Due do the invasive nature of Harding grass, we sought to examine whether it has an effect on small animal abundance and diversity in the grasslands, Rancho Marino Reserve (RM) and Fiscalini Ranch Preserve (FR) in California. Both grasslands have similar climate and geographic location but differ in management history. Two transects were created in each site, with eight plots per transect. Animal cameras were deployed over the course of three nights to examine the abundance and diversity of small animals. Due to the history of tilling and planting of RM, and its increase in P. aquatica coverage, there was less animal abundance and diversity compared to FR. The results indicated that the untilled/unplanted areas had more animal abundance and diversity compared to tilled/planted due to the lack of Harding Grass. This can be due to factors such as diminished soil quality, difficulty in maneuvering in the tall grass, and adaptability to native vegetative state. Invasive plants have the ability to increase rapidly in space and potentially lead to ecosystem degradation. This adds further knowledge in the relationship between small animals and their habitats and helps conservation biologists ensure mammalian populations remain stable.

Effect of Phalaris aquatica on the abundance and diversity of vertebrate animals in Mediterranean coastal grasslands in California

Harding grass (Phalaris aquatica), an invasive non-native species of bunchgrass, has been introduced to grasslands in many regions of California, particularly those with a history of disturbance, such as tilling and grazing. Due do the invasive nature of Harding grass, we sought to examine whether it has an effect on small animal abundance and diversity in the grasslands, Rancho Marino Reserve (RM) and Fiscalini Ranch Preserve (FR) in California. Both grasslands have similar climate and geographic location but differ in management history. Two transects were created in each site, with eight plots per transect. Animal cameras were deployed over the course of three nights to examine the abundance and diversity of small animals. Due to the history of tilling and planting of RM, and its increase in P. aquatica coverage, there was less animal abundance and diversity compared to FR. The results indicated that the untilled/unplanted areas had more animal abundance and diversity compared to tilled/planted due to the lack of Harding Grass. This can be due to factors such as diminished soil quality, difficulty in maneuvering in the tall grass, and adaptability to native vegetative state. Invasive plants have the ability to increase rapidly in space and potentially lead to ecosystem degradation. This adds further knowledge in the relationship between small animals and their habitats and helps conservation biologists ensure mammalian populations remain stable.

Persistence and productivity of phalaris (Phalaris aquatica) germplasm in dry marginal rainfall environments of south-eastern Australia

Perennial grasses have production and environmental benefits in areas of southern Australia typified by the mixed farming zone of southern New South Wales (NSW). The perennial grass phalaris (Phalaris aquatica L.) is widely used in southern Australia; however, it would find more use in the mixed farming zone if its persistence in marginal rainfall areas (450–500 mm average annual rainfall) were improved. We evaluated a range of germplasm (n = 29) including wild accessions, lines bred from these, and existing cultivars for persistence and production at three sites in a summer-dry area of southern NSW with 430–460-mm average annual rainfall. Two sites were used over 4 years and the third site over 5 years. Summer dormancy, maturity time and seedling growth were also assessed. Analysis of genotype × environment interaction employing factor analytic models and accounting for spatial and temporal correlations indicated that changes in persistence occurred mainly over time rather than between sites. Ranking changes occurred in the dry establishment phase of the experiment and during a severe final summer drought, with few changes occurring in the intervening high-rainfall years. Lines that survived the establishment phase best had vigorous seedlings and earlier maturity, whereas those surviving the final summer best were earlier maturing and higher in summer dormancy with high winter-growth activity. Some later maturing lines within the higher summer dormancy group were less persistent. Some accessions from North Africa were the most persistent; also, populations bred from these and other more persistent accessions generally persisted and produced better than cultivars used presently. However, present cultivars were capable of high yield in the higher rainfall years. We suggest that persistence of higher summer dormancy cultivars over very dry years could be improved by selecting for earlier maturity time.