Volume 66, Issue 7 p. 1162-1181
Original Article
Free Access

Severity of phytophthora root rot and pre-emergence damping-off in subterranean clover influenced by moisture, temperature, nutrition, soil type, cultivar and their interactions

M. P. You

M. P. You

School of Agriculture and Environment and the UWA Institute of Agriculture, Faculty of Science, The University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009 Australia

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M. J. Barbetti

Corresponding Author

M. J. Barbetti

School of Agriculture and Environment and the UWA Institute of Agriculture, Faculty of Science, The University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009 Australia

E-mail: [email protected]Search for more papers by this author
First published: 29 November 2016
Citations: 12

Abstract

Studies were carried out in controlled environment rooms reflecting field situations. In the presence of the devastating soilborne pathogen Phytophthora clandestina, subterranean clover (Trifolium subterraneum) seedling emergence was significantly affected by moisture, soil type, temperature and cultivar. The level of rotting of tap and lateral roots was significantly affected by nutrition, soil type, temperature and cultivar. There were significant interactions involving temperature, moisture, soil type and cultivar; cultivar resistance, high moisture, high or medium temperature, high nutrition and sand soil all contributed towards less pre-emergence damping-off and tap and lateral root disease and to greater clover productivity. Host resistance of subterranean clover cultivars was critical for reducing disease severity and increasing productivity, even when favourable environmental conditions for severe disease occurred. In the presence of P. clandestina, the most resistant cultivar, Seaton Park, performed best under a high temperature, high nutrition and high moisture combination, but showed lower productivity under conditions of low nutrition or lower temperature, even when moisture level was high. In contrast, less resistant cultivars Riverina and Meteora had less disease and greater productivity under low moisture conditions. Findings reflect field observations that pre-emergence damping-off and root disease from P. clandestina in subterranean clover is particularly severe under colder conditions and in nutritionally impoverished sandy soils, and demonstrate how variations in soil type, nutrition, moisture, temperature and cultivar have profound effects on the expression and severity of phytophthora pre-emergence damping-off and root disease and the productivity of subterranean clover forages.

Introduction

Subterranean clover (Trifolium subterraneum) is an important component of agronomic systems worldwide, including regions with Mediterranean-type climates in Africa, Asia, Australia, Europe, North America and South America (Nichols et al., 2014). It is particularly important in southern Australia where it has been grown on 29 million ha (Hill & Donald, 1998). Subterranean clover provides a valuable source of nutritious livestock feed and of nitrogen for cereal crops. However, subterranean clover is attacked by a diverse range of soilborne pathogens, especially oomycetes. These soilborne pathogens cause severe pre-emergence damping-off and root disease in seedlings (Wong et al., 1984; Barbetti et al., 1986a,b, 2007) and root disease in mature subterranean clover swards (O'Rourke et al., 2009), resulting in severe reduction of forage (Gillespie, 1983; Barbetti et al., 1986a). Widespread decline and failure of persistence of subterranean clover forages leads to serious reductions in livestock carrying capacity and whole farm profitability across southern Australia (Barbetti et al., 1986b; Nichols et al., 2014). This decline not only manifests as a decrease in the desirable legume component, but also as an increase in weeds (M. J. Barbetti, unpublished data).

The most important soilborne pathogens of subterranean clover include Phytophthora clandestina, various Pythium species such as Pythium irregulare, Aphanomyces trifolii and Rhizoctonia solani (Wong et al., 1985; Barbetti et al., 2006, 2007; Ma et al., 2008; Nichols et al., 2014). Of these, P. clandestina is generally considered the most important (You et al., 2005a,b; Barbetti et al., 2007), and it is also known to affect other forage legumes, such as Biserrula pelecinus, Hedysarum coronarium, Ornithopus compressus and O. sativus (Li et al., 2009).

Phytophthora clandestina was first detected on rotted tap roots of subterranean clover in Victoria, Australia, in 1982 (Taylor, 1984; Greenhalgh & Taylor, 1985; Taylor et al., 1985c). Greenhalgh (1992) estimated that this pathogen could reduce annual production of subterranean clover by >90%. Severe disease particularly occurred in high rainfall areas and seasons and in irrigated areas (Taylor et al., 1985b; Greenhalgh & Flett, 1987; Taylor & Greenhalgh, 1987). Phytophthora clandestina is also an important root pathogen of subterranean clover in New South Wales, Australia (Dear et al., 1993) and in South Australia (M. P. You, unpublished data). In Western Australia, the presence of P. clandestina was confirmed in 1984 (Taylor et al., 1985a) and recognized as one of the most serious pathogens of the soilborne pathogen complex there (Wong et al., 1985, 1986c). Subsequently, You et al. (2005b) characterized a total of 10 races of P. clandestina in Western Australia, of which races 173 and 177 were widely distributed and common in Western Australia, together constituting 80% of isolates characterized.

There have been a number of historical attempts to relate environmental factors to the severity of root disease. For example, analysis of climatic data for centres along the south coast of Western Australia from 1972 to 1975 showed that in 1973, a particularly severe root disease year, the Mediterranean ecosystem of southwest Western Australia had significantly heavier and more frequent rainfall after the ‘opening’ seasonal rains in autumn than in other years when root disease severity was much lower (MacNish et al., 1976). This is not surprising, as wet soil conditions strongly favour oomycete pathogens such as P. clandestina, A. trifolii and various Pythium spp. There have also been some specific studies of environmental effects on the behaviour of this pathogen; for example, Wong et al. (1986b,d) investigated the soil behaviour of this pathogen in the field, and also investigated the influence of some individual environmental factors on the growth and survival of P. clandestina (Wong et al., 1986c,e). On occasion, there has been investigation of the interaction of different environmental factors, but only restricted to a maximum of two factors. For example, Wong et al. (1986c) described the interactions of P. clandestina with soil temperature and moisture and showed that the most severe root disease occurred at a soil temperature of 10 °C, followed by less severe root disease at 15 °C than at 20 °C, coinciding with autumn/early winter conditions. They also showed that the greatest reductions in seedling survival occurred in saturated and flooded soil. Further, and subsequent to the above studies for soilborne diseases of subterranean clover, there have been strong indications that both soil type (M. J. Barbetti, unpublished data) and nutrition (O'Rourke et al., 2012) may also play a significant role in the expression of root disease. Previous studies were not able to explain the wide variations in disease severity and above-ground symptoms observed across southern Australia, perhaps because too few environmental factors were considered in each individual study. Hence, the present investigation was undertaken to define the individual and interactive effects of temperature, moisture, soil type, nutrition and cultivar on the severity of phytophthora pre-emergence damping-off and root rot and on plant productivity, using environmental conditions representative of the variation across southern Australian subterranean clover forages.

Materials and methods

Subterranean clover cultivars

Cultivars Meteora, Riverina, Seaton Park and Woogenellup were used in this experiment as they each have different levels of resistance to P. clandestina. Seaton Park is known to have the greatest resistance to the majority of known races, compared with other cultivars. Woogenellup is susceptible to nearly all races of P. clandestina (You et al., 2005b) and is also used as the universal susceptible control comparison for all soilborne root disease studies on subterranean clover (Barbetti et al., 2007). Subterranean clover seeds were surface sterilized in 70% ethanol for 30 s to remove any seed pathogen contamination; they were then scarified lightly with sandpaper to break dormancy and increase germination and sown at five seeds per pot at a depth of 10 mm. Pots were 9 × 9 cm square with a 10 cm depth.

Temperature regimes

Experiments were conducted in three separate controlled environment rooms with temperatures maintained at 22/17 °C, 18/13 °C or 14/9 °C (day/night) with a 12 h photoperiod and light intensity of 480–572 μmol m−2 s−1. These temperatures were selected to mimic temperatures commonly seen in the field in Western Australia during May–August when root disease is prevalent in subterranean clover forages (Barbetti, 1991).

Moisture levels

There were two levels of moisture; high moisture pots were watered to free draining with deionized water (DI) water daily (i.e. to 100% water holding capacity (WHC)), and water was added to low moisture pots as required (e.g. every second day on high temperature pots) to maintain 50% WHC.

Soil types and treatments

Two types of soil were used; a sand-based mix representing light soil type (airing) and a Gingin red loam soil representing a heavier soil type (compact). The sand-based mix soil consisted of 2.5 m3 fine composted pine bark, 1 m3 coco peat, 5 m3 brown washed river sand, 10 kg slow release fertilizer Osmoform NXT 22 N + 2.2 P2O5 + 9.1 K2O + 1.2 Mg + trace elements (Everris International B.V.), 10 kg dolomite (CalMag), 5 kg gypsum clay breaker, 5 kg extra fine limestone, 4 kg iron heptasulphate and 1 kg iron chelate. The Gingin red was a loam soil with a sand content of 85% (w/w) (McArthur, 1991); this soil has a texture that relates to extensive soil areas within and outside Western Australia. Each soil was pasteurized using aerated steam at 65 °C for 90 min on each of three consecutive days prior to use.

Nutrition levels and soil nutrient analyses

Two levels of nutrition were used, viz. high nutrition, where seedlings were fertilized weekly, at the recommended rate, with a complete range of nutrients (Thrive; Yates) required for seedling growth and development, and low nutrition treatment where seedlings were watered with only DI water throughout the experiment. Pooled soil samples collected from root zones around subterranean clover seedlings from the same treatment were air dried at 25–30 °C in a glasshouse, and sent to CSBP Plant and Soil Analysis Ltd, Western Australia for nutrient analyses. Characteristics, including ammonium-N, nitrate-N, available-P, K and S, organic carbon, conductivity, pH (CaCl2), pH (H2O), DTPA Cu, Fe, Mn and Zn, exchangeable Al, Ca, Mg, K and Na, boron hot CaCl2, total N, P and K, were assessed using the protocols outlined by O'Rourke et al. (2012). For P levels, the Colwell-P (the labile P pool, easily available to the plant) was measured (Colwell, 1963, 1965), while for Cu, Fe, Zn and Mn, an extractable soil test using diethylenetriaminepentaacetic acid (DTPA) was used.

The nutrient status of high versus low nutrition soils at the harvest time is shown in Table 1. In summary, in comparison with the Gingin red loam, sand-based mix under high nutrient treatment contained higher nitrate nitrogen (15.56 mg kg−1), phosphorus (Colwell, 39.67 mg kg−1), potassium (Colwell, 79.72 mg kg−1), sulphur (28.8 mg kg−1), organic carbon (4.22%), conductivity (0.11 dS m−1), copper (0.71 mg kg−1), iron (17.27 mg kg−1), zinc (1.65 mg kg−1), potassium (89.33 meq/100 g), sodium (0.07 meq/100 g), boron (0.38 mg kg−1), nitrogen (total, 0.09%), phosphorus (total, 79.95 mg kg−1) and potassium (total, 89.33 mg kg−1). Under low nutrition treatment, in comparison with the Gingin red loam, the sand-base mix contained the highest levels of manganese (2.34 mg kg−1), calcium (6.321 meq/100 g), and exchangeable magnesium (2.34 meq/100 g). Exceptions to the above were where Gingin red loam under high nutrition treatment contained higher levels of ammonium nitrogen (11.06 mg kg−1) and under both low and high nutrition contained higher aluminium (0.06 and 0.05 meq/100 g, respectively) in comparison with the sand-based mix.

Table 1. Summary of main nutrition components and their levels in sandy versus loamy soils under ‘high’ and ‘low’ nutritional regimes
Component High nutrition Low nutrition
Sand Loam Sand Loam
Nitrate nitrogen (mg kg−1) 15.556 (1)a 0.050 (4) 7.611 (2) 0.467 (3)
Phosphorus Colwellb (mg kg−1) 39.667 (1) 8.611 (3) 32.389 (2) 4.333 (4)
Potassium Colwell (mg kg−1) 79.722 (1) 19.222 (3) 67.111 (2) 14.278 (4)
Sulphur (mg kg−1) 28.800 (1) 3.044 (3) 27.700 (2) 2.011 (4)
Organic carbon (%) 4.223 (1) 0.157 (4) 4.029 (2) 0.165 (3)
Conductivity (dS m−1) 0.112 (1) 0.020 (3) 0.089 (2) 0.013 (4)
DTPAc copper (mg kg−1) 0.706 (1) 0.127 (4) 0.619 (2) 0.132 (3)
DTPA iron (mg kg−1) 17.269 (1) 5.653 (3) 17.098 (2) 5.482 (4)
DTPA zinc (mg kg−1) 1.653 (1) 0.045 (3) 1.567 (2) 0.031 (4)
Exc.d potassium (meq/100 g) 0.205 (1) 0.050 (3) 0.171 (2) 0.030 (4)
Exc. sodium (meq/100 g) 0.069 (1) 0.013 (3) 0.064 (2) 0.012 (4)
Boron hot CaCl2 (mg kg−1) 0.379 (1) 0.133 (4) 0.332 (2) 0.136 (3)
Total nitrogen (%) 0.095 (1) 0.008 (4) 0.088 (2) 0.009 (3)
Total phosphorus (mg kg−1) 79.950 (1) 73.994 (3) 75.228 (2) 65.172 (4)
Total potassium (mg kg−1) 89.333 (1) 38.889 (3) 82.222 (2) 30.889 (4)
Ammonium nitrogen (mg kg−1) 6.033 (2) 11.056 (1) 2.978 (4) 3.022 (3)
DTPA manganese (mg kg−1) 2.326 (2) 0.119 (4) 2.336 (1) 0.143 (3)
Exc. calcium (meq/100 g) 6.157 (2) 0.302 (4) 6.321 (1) 0.312 (3)
Exc. magnesium (meq/100 g) 1.311 (2) 0.068 (3) 1.329 (1) 0.065 (4)
Exc. aluminium (meq/100 g) 0.007 (3) 0.046 (2) 0.007 (4) 0.065 (1)
pH level (CaCl2) 5.283 (4) 5.967 (1) 5.422 (3) 5.656 (2)
pH level (H2O) 6.250 (4) 6.667 (1) 6.372 (3) 6.433 (2)
  • Samples were taken at the time of harvesting plants for disease and other assessments.
  • a Numbers in brackets represent the relative ranking across the four combinations of high nutrition level and sandy soil, high nutrition and loamy soil, low nutrition and sandy soil and low nutrition and loamy soil. (1) indicates the highest comparative level and (4) the lowest comparative level.
  • b Colwell, Colwell-P by measuring the labile, easily plant-available P pool.
  • c DTPA, DTPA-extractable soil test using diethylenetriaminepentaacetic acid.
  • d Exc., exchangeable.

Isolation of P. clandestina

Isolates of P. clandestina were obtained from infested subterranean clover soils sampled from a region on the south coast of Western Australia, where phytophthora root disease and damping-off are prevalent. This was achieved using subterranean clover cv. Woogenellup as the ‘bait species’. In brief, cv. Woogenellup plants were grown in infested soil samples in a controlled environment room at 18/13 °C (day/night), with a 12/12 h photoperiod; then pots were flooded for 1 h at 1 and 3 weeks after sowing, and harvested at 4 weeks. Harvested plant roots were thoroughly washed under running tap water to remove soil. Whole root systems were floated in Petri dishes containing sterile distilled water and maintained in the dark in an incubator at 20 °C. Using a light microscope, individual P. clandestina zoosporangia were collected (at 24–48 h), using fine-tip tweezers, and placed directly onto Petri dishes containing a modified metalaxyl-benomyl-vancomycin agar (MBV) agar (Pfender et al., 1984). Phytophthora clandestina cultures were then subcultured onto fresh lima bean agar (LBA) and grown for 1 week at 25 °C under neon lighting in preparation for inoculum production. One isolate of P. clandestina, identified as race 173, was selected for these studies by testing isolates across the seven standard subterranean clover host differentials (You et al., 2005b). This race was chosen because it is the prevailing race on subterranean clover in southwest Western Australia (You et al., 2005b).

Inoculum production of P. clandestina

Inoculum was prepared using a modified procedure from Barbetti (1989). In brief, 2-week-old P. clandestina colonies growing on LBA were cut into plugs 2 mm2 and approximately 10 plugs were used to inoculate each 250 mL Erlenmeyer flask containing sterilized millet seeds (Panicum miliaceum). Millet seeds were prepared by soaking 100 g of seeds in 100 mL DI water overnight in each 250 mL Erlenmeyer flask and then the water was drained and the flasks containing the seeds were autoclaved at 120 °C for 20 min, three times on each of three consecutive days. Every second day, flasks with inoculated millet seeds were hand shaken vigorously to homogenize the inoculum with the millet seeds to ensure equal colonization. Inoculated millet seeds were incubated at 22 °C for 3 weeks. Colonized millet seeds were then used for inoculating soils. At the time of inoculation, millet seed inoculum was also replated onto LBA to confirm that P. clandestina was present.

Inoculation of soil with P. clandestina

One half of each soil type was mixed thoroughly with P. clandestina-colonized millet seeds at a rate of 0.5% (w/w) and used to fill pots. As a control, pots were filled with uninfested soil of each type, but no uncolonized millet seeds were added as uncolonized millet can readily ‘bait-out’ other, non-target, soilborne pathogens present (Barbetti & Sivasithamparam, 1987). Seeds of subterranean clover were sown in the inoculated and noninoculated pots, as described above, and the pots were flooded separately for 1 h, immediately after sowing, with flooding repeated 2 weeks later. At 4 weeks after sowing, pots were flooded again for 2 h.

Disease, nodulation and plant weight assessments

Germinated plants in each pot were counted to calculate the percentage of emergence before harvesting. Then, plants were harvested at 5 weeks after sowing, washed in running tap water to remove soil from roots, and scored for their level of root disease. Subsequently, plants were floated in shallow trays of DI water and both tap and lateral roots were visually scored independently using a modified scoring system described by Wong et al. (1984). This assessment scale contained six disease severity categories: score 0 = root healthy, no discolouration; 1 = <25% of root brown, no significant lesions; 2 = 25–49% of root brown, lesions towards base of tap root; 3 = 50–74% root brown, lesions mid tap root; 4 = ≥75% root affected, significant lesions towards crown; 5 = plant dead and/or root system completely rotted off. The number of plants in each disease severity category was recorded. Then, all disease rating scores were transferred to a tap or lateral percentage disease index (PDI) based on McKinney (1923), where tap or lateral root PDI = (sum of all numerical grades) × 100 ÷ (total number of plants scored × maximum rating score).

At the same time, plants were also assessed for level of nodulation on roots, using a modified rating scheme from Corbin et al. (1977), with a 0 to 5 scale where: 0 = no nodules on the crown or elsewhere; 1 = no nodules on the crown with a few (1–10) elsewhere; 2 = a few crown nodules but no nodules elsewhere; 3 = many crown nodules (>10) but no nodules elsewhere; 4 = many crown nodules with a few nodules elsewhere; 5 = many crown nodules with many nodules elsewhere. All nodulation rating scores were transferred to a percentage nodulation index (PNI) based on McKinney (1923), where PNI = (sum of all numerical grades) × 100 ÷ (total number of plants scored × maximum rating score).

Shoots and roots from each pot were separated and dried at 60 °C in an oven in separate paper bags for 3 days. Then, dry shoot and root weights were recorded and calculated as mg per plant.

Confirmation of presence of P. clandestina

In all experiments Koch's postulates were successfully completed to confirm that the disease symptoms observed were in fact caused by P. clandestina. Root segments (8–10), 2 cm in length, were dissected from diseased plants and floated in Petri dishes containing sterile DI water for 2–3 days at 20 °C. Roots were examined microscopically every 12 h using a light microscope and the presence of P. clandestina zoosporangia was confirmed.

Experimental design and analyses

There were four replicate pots for each treatment, with treatments in a factorial arrangement, and all pots were maintained in their respective temperature-controlled environmental rooms throughout. All inoculated treatments were repeated using noninoculated soils as a control. This experiment was arranged in a randomized block design and the whole experiment for inoculated and noninoculated soils was repeated once under the same conditions.

Analysis of variance (anova) was conducted using GenStat (14th edition; Lawes Agricultural Trust). Normality of data and homogeneity of variances from each experiment were tested before conducting analyses. Data from the original and the repeat experiments were not significantly different (> 0.05) using a t-test. Therefore, data from the original and repeat experiments were combined and reanalysed together. Data on emergence, disease index of tap roots, disease index of lateral roots, dry root weight (DRW) and dry shoot weight (DSW) of seedlings from each experiment had a normal distribution and similar variance. Therefore, the effects of different treatments (i.e. temperature, soil type, moisture and nutrition, cultivars and pathogen inoculation) on emergence, tap root disease, lateral root disease, DRW and DSW of seedlings were determined by analyses of variance, and subsequent multiple comparisons between treatments were made using Fisher's protected least significant differences (l.s.d.) at = 0.05. Standard errors (SE) of means were also computed. GenStat was also used to test the significance of correlation coefficients between the parameters assessed and the different treatments used.

Results

Emergence

In the presence of P. clandestina under the conditions of low, medium and high temperature regimes (14/9 °C, 18/13 °C, and 22/17 °C day/night), high and low moisture, high and low nutrition and two soil types (sand and loam sand), percentage seedling emergence (i.e. survival) was significantly affected by single factors of moisture, pathogen, soil type, temperature and cultivar (Tables 2 & 3). In brief, percentage emergence was greatest under high moisture, in sand soil, at medium to high temperatures and, as expected, in the absence of P. clandestina. Cultivar Seaton Park had the greatest percentage emergence followed by Meteora and Riverina, while Woogenellup had the lowest percentage emergence. Factors affecting percentage emergence and their interactions are detailed in Figure 1.

Table 2. Statistical main effects and interactions of environmental factors on disease indices (%) of tap and lateral root rot, nodulation index (%), emergence (%), and dry weights (mg per plant) of root and shoot
Factor TDI% LDI% NI% E% DRW (mg per plant) DSW (mg per plant)
P LSD0.05 P LSD0.05 P LSD0.05 P LSD0.05 P LSD0.05 P LSD0.05
Single factors
Moisture Ns 0.985 <0.001 0.831 Ns 0.1738 0.006 2.045 <0.001 2.019 Ns 12.91
Nutrition <0.001 0.985 <0.001 0.831 <0.001 0.1738 Ns 2.045 <0.001 2.019 <0.001 12.91
Pathogen <0.001 0.985 <0.001 0.831 <0.001 0.1738 0.036 2.045 <0.001 2.019 <0.001 12.91
Soil <0.001 0.985 <0.001 0.831 <0.001 0.1738 <0.001 2.045 <0.001 2.019 <0.001 12.91
Temperature <0.001 1.207 <0.001 1.018 <0.001 0.2129 <0.001 2.504 <0.001 2.473 <0.001 15.81
Cultivar <0.001 1.393 <0.001 1.175 <0.001 0.2458 <0.001 2.892 0.036 2.855 0.005 18.26
Interactions
Moisture × nutrition Ns 1.393 Ns 1.175 Ns 0.2458 Ns 2.892 Ns 2.855 Ns 18.26
Moisture × pathogen Ns 1.393 <0.001 1.175 Ns 0.2458 0.048 2.892 <0.001 2.855 Ns 18.26
Nutrition × pathogen <0.001 1.393 <0.001 1.175 <0.001 0.2458 Ns 2.892 Ns 2.855 0.003 18.26
Moisture × soil <0.001 1.393 <0.001 1.175 Ns 0.2458 <0.001 2.892 Ns 2.855 Ns 18.26
Nutrition × soil Ns 1.393 Ns 1.175 <0.001 0.2458 Ns 2.892 0.008 2.855 Ns 18.26
Pathogen × soil <0.001 1.393 <0.001 1.175 <0.001 0.2458 0.009 2.892 Ns 2.855 <0.001 18.26
Moisture × temperature Ns 1.706 Ns 1.44 Ns 0.301 <0.001 3.542 0.002 3.497 0.002 22.37
Nutrition × temperature 0.002 1.706 0.002 1.44 <0.001 0.301 Ns 3.542 <0.001 3.497 0.035 22.37
Pathogen × temperature <0.001 1.706 <0.001 1.44 <0.001 0.301 Ns 3.542 <0.001 3.497 0.037 22.37
Soil × temperature <0.001 1.706 <0.001 1.44 <0.001 0.301 <0.001 3.542 Ns 3.497 0.025 22.37
Moisture × cultivar Ns 1.97 0.014 1.662 <0.001 0.3476 <0.001 4.09 0.016 4.038 Ns 25.83
Nutrition × cultivar Ns 1.97 Ns 1.662 <0.001 0.3476 Ns 4.09 Ns 4.038 Ns 25.83
Pathogen × cultivar <0.001 1.97 <0.001 1.662 <0.001 0.3476 <0.001 4.09 <0.001 4.038 <0.001 25.83
Soil × cultivar <0.001 1.97 <0.001 1.662 <0.001 0.3476 Ns 4.09 0.003 4.038 Ns 25.83
Temperature × cultivar <0.001 2.413 <0.001 2.036 <0.001 0.4257 <0.001 5.009 <0.001 4.945 Ns 31.63
Moisture × nutrition × pathogen Ns 1.97 Ns 1.662 Ns 0.3476 Ns 4.09 0.024 4.038 Ns 25.83
Moisture × nutrition × soil Ns 1.97 0.048 1.662 Ns 0.3476 Ns 4.09 Ns 4.038 Ns 25.83
Moisture × pathogen × soil <0.001 1.97 <0.001 1.662 Ns 0.3476 Ns 4.09 Ns 4.038 Ns 25.83
Nutrition × pathogen × soil Ns 1.97 Ns 1.662 <0.001 0.3476 Ns 4.09 Ns 4.038 Ns 25.83
Moisture × nutrition × temperature Ns 2.413 Ns 2.036 Ns 0.4257 Ns 5.009 0.008 4.945 0.029 31.63
Moisture × pathogen × temperature Ns 2.413 Ns 2.036 Ns 0.4257 Ns 5.009 Ns 4.945 Ns 31.63
Nutrition × pathogen × temperature 0.002 2.413 0.002 2.036 <0.001 0.4257 Ns 5.009 <0.001 4.945 Ns 31.63
Moisture × soil × temperature 0.047 2.413 Ns 2.036 Ns 0.4257 Ns 5.009 Ns 4.945 Ns 31.63
Nutrition × soil × temperature Ns 2.413 Ns 2.036 <0.001 0.4257 Ns 5.009 0.029 4.945 Ns 31.63
Pathogen × soil × temperature <0.001 2.413 <0.001 2.036 <0.001 0.4257 0.02 5.009 Ns 4.945 Ns 31.63
Moisture × nutrition × cultivar <0.001 2.787 <0.001 2.351 Ns 0.4916 Ns 5.784 Ns 5.71 Ns 36.52
Moisture × pathogen × cultivar Ns 2.787 Ns 2.351 <0.001 0.4916 0.045 5.784 Ns 5.71 Ns 36.52
Nutrition × pathogen × cultivar Ns 2.787 Ns 2.351 <0.001 0.4916 Ns 5.784 Ns 5.71 Ns 36.52
Moisture × soil × cultivar Ns 2.787 <0.001 2.351 <0.001 0.4916 Ns 5.784 Ns 5.71 Ns 36.52
Nutrition × soil × cultivar 0.04 2.787 0.006 2.351 <0.001 0.4916 Ns 5.784 Ns 5.71 Ns 36.52
Pathogen × soil × cultivar <0.001 2.787 <0.001 2.351 <0.001 0.4916 Ns 5.784 <0.001 5.71 Ns 36.52
Moisture × temperature × cultivar Ns 3.413 <0.001 2.879 <0.001 0.602 0.012 7.083 Ns 6.994 Ns 44.73
Nutrition × temperature × cultivar Ns 3.413 0.002 2.879 <0.001 0.602 Ns 7.083 Ns 6.994 Ns 44.73
Pathogen × temperature × cultivar <0.001 3.413 <0.001 2.879 <0.001 0.602 Ns 7.083 0.015 6.994 <0.001 44.73
Soil × temperature × cultivar <0.001 3.413 Ns 2.879 <0.001 0.602 0.003 7.083 0.004 6.994 Ns 44.73
Moisture × nutrition × pathogen × soil Ns 2.787 Ns 2.351 Ns 0.4916 Ns 5.784 Ns 5.71 Ns 36.52
Moisture × nutrition × pathogen × temperature Ns 3.413 Ns 2.879 Ns 0.602 Ns 7.083 <0.001 6.994 Ns 44.73
Moisture × nutrition × soil × temperature Ns 3.413 <0.001 2.879 Ns 0.602 0.005 7.083 Ns 6.994 Ns 44.73
Moisture × pathogen × soil × temperature 0.042 3.413 Ns 2.879 Ns 0.602 Ns 7.083 Ns 6.994 Ns 44.73
Nutrition × pathogen × soil × temperature Ns 3.413 Ns 2.879 <0.001 0.602 Ns 7.083 Ns 6.994 Ns 44.73
Moisture × nutrition × pathogen × cultivar <0.001 3.941 <0.001 3.325 Ns 0.6952 Ns 8.179 Ns 8.076 Ns 51.65
Moisture × nutrition × soil × cultivar <0.001 3.941 <0.001 3.325 0.016 0.6952 Ns 8.179 Ns 8.076 Ns 51.65
Moisture × pathogen × soil × cultivar Ns 3.941 <0.001 3.325 <0.001 0.6952 0.004 8.179 0.007 8.076 Ns 51.65
Nutrition × pathogen × soil × cultivar 0.038 3.941 0.005 3.325 <0.001 0.6952 Ns 8.179 Ns 8.076 Ns 51.65
Moisture × nutrition × temperature × cultivar 0.043 4.827 0.04 4.072 0.006 0.8514 Ns 10.018 Ns 9.89 Ns 63.26
Moisture × pathogen × temperature × cultivar Ns 4.827 <0.001 4.072 <0.001 0.8514 0.02 10.018 Ns 9.89 Ns 63.26
Nutrition × pathogen × temperature × cultivar Ns 4.827 0.001 4.072 <0.001 0.8514 Ns 10.018 Ns 9.89 Ns 63.26
Moisture × soil × temperature × cultivar 0.023 4.827 0.006 4.072 <0.001 0.8514 0.024 10.018 Ns 9.89 Ns 63.26
Nutrition × soil × temperature × cultivar Ns 4.827 Ns 4.072 <0.001 0.8514 Ns 10.018 0.032 9.89 Ns 63.26
Pathogen × soil × temperature × cultivar <0.001 4.827 Ns 4.072 <0.001 0.8514 0.047 10.018 Ns 9.89 Ns 63.26
Moisture × nutrition × pathogen × soil × temperature Ns 4.827 <0.001 4.072 Ns 0.8514 Ns 10.018 0.022 9.89 Ns 63.26
Moisture × nutrition × pathogen × soil × cultivar <0.001 5.573 <0.001 4.702 0.016 0.9831 Ns 11.567 Ns 11.421 Ns 73.05
Moisture × nutrition × pathogen × temperature × cultivar 0.045 6.826 0.045 5.759 0.006 1.2041 Ns 14.167 0.005 13.987 Ns 89.46
Moisture × nutrition × soil × temperature × cultivar <0.001 6.826 <0.001 5.759 0.016 1.2041 Ns 14.167 Ns 13.987 Ns 89.46
Moisture × pathogen × soil × temperature × cultivar 0.021 6.826 0.006 5.759 <0.001 1.2041 Ns 14.167 Ns 13.987 Ns 89.46
Nutrition × pathogen × soil × temperature × cultivar Ns 6.826 Ns 5.759 <0.001 1.2041 Ns 14.167 Ns 13.987 Ns 89.46
Moisture × nutrition × pathogen × soil × temperature × cultivar <0.001 9.653 <0.001 8.144 0.019 1.7028 Ns 20.035 Ns 19.781 Ns 126.52
  • TDI, tap root disease index (%); LDI, lateral root disease index (%); NI, nodulation index (%); E, emergence (%); DRW, dry root weight (mg per plant); and DSW, dry shoot weight (mg per plant). Ns, not significant, P > 0.05.
Table 3. Effects of environmental factors on disease indices (%) of tap and lateral roots, nodulation index (%), emergence (%), dry weights of root and shoot (mg per plant)
Moisture Nutrition Pathogen Soil type Temperature Cultivar Mean
Meteora Riverina Seaton Park Woogenellup
Tap root disease index % High High Phytophthora Loam High 41.08 30.46 20.54 80.07 43.04
Medium 27.87 29.79 19.33 85.00 40.50
Low 55.04 55.83 26.21 86.75 55.96
Sand High 27.42 23.79 3.33 78.24 33.20
Medium 50.25 46.83 11.63 82.50 47.80
Low 41.67 45.83 4.58 70.12 40.55
Low Phytophthora Loam High 34.79 33.54 8.54 39.86 29.18
Medium 35.37 37.50 17.83 85.00 43.93
Low 49.87 49.38 4.63 78.74 45.66
Sand High 19.75 22.71 0.50 76.11 29.77
Medium 36.00 37.42 15.50 77.50 41.61
Low 27.79 32.79 2.38 59.81 30.69
Low High Phytophthora Loam High 48.04 58.05 25.37 50.53 45.50
Medium 41.67 51.67 32.71 87.50 53.39
Low 63.33 59.17 50.42 83.23 64.04
Sand High 16.42 24.58 3.08 75.28 29.84
Medium 21.33 35.00 12.00 76.25 36.15
Low 29.79 49.50 3.13 70.40 38.21
Low Phytophthora Loam High 38.43 30.33 14.25 84.99 42.00
Medium 45.75 36.00 15.58 87.50 46.21
Low 47.50 44.58 18.00 73.33 45.85
Sand High 19.75 28.40 0.00 66.89 28.76
Medium 23.12 20.83 0.00 87.50 32.86
Low 23.54 35.58 0.63 59.81 29.89
Lateral root disease index % High High Phytophthora Loam High 14.79 15.46 6.58 80.32 29.29
Medium 12.87 26.46 8.00 85.00 33.08
Low 49.77 57.92 7.29 79.99 48.74
Sand High 13.79 17.62 4.62 76.86 28.22
Medium 29.46 44.17 4.62 87.50 41.44
Low 47.63 48.54 1.88 79.85 44.48
Low Phytophthora Loam High 19.17 20.62 2.17 39.97 20.48
Medium 36.71 33.33 8.33 87.50 41.47
Low 41.25 52.71 2.50 68.82 41.32
Sand High 17.50 17.00 0.00 70.53 26.26
Medium 30.00 31.21 6.50 77.50 36.30
Low 28.88 47.92 1.25 79.82 39.47
Low High Phytophthora Loam High 40.58 57.35 27.54 50.16 43.91
Medium 54.17 66.46 33.25 90.00 60.97
Low 70.00 66.04 50.12 83.25 67.35
Sand High 13.92 14.58 0.00 75.47 25.99
Medium 15.54 19.58 1.62 77.50 28.56
Low 32.50 35.50 0.00 80.02 37.01
Low Phytophthora Loam High 35.84 33.00 4.50 87.74 40.27
Medium 45.92 51.17 11.96 87.50 49.14
Low 56.25 60.42 25.12 79.97 55.44
Sand High 18.00 15.61 0.00 55.23 22.21
Medium 23.75 13.33 0.00 87.50 31.15
Low 12.54 17.25 0.00 76.01 26.45
Emergence rate % High High Nil Loam High 70.00 62.50 80.00 28.56 60.27
Medium 65.00 60.00 65.00 17.50 51.88
Low 52.50 67.50 35.00 20.00 43.75
Sand High 75.00 57.50 85.00 47.50 66.25
Medium 82.50 62.50 95.00 35.00 68.75
Low 70.00 65.00 70.00 30.00 58.75
Phytophthora Loam High 72.50 72.50 85.00 21.39 62.85
Medium 70.00 45.00 90.00 17.50 55.63
Low 52.50 57.50 67.50 12.50 47.50
Sand High 72.50 65.00 85.00 34.82 64.33
Medium 80.00 65.00 77.50 20.00 60.63
Low 72.50 62.50 82.50 17.50 58.75
Low Nil Loam High 72.50 60.00 85.00 40.00 64.38
Medium 65.00 47.50 82.50 35.00 57.50
Low 65.00 47.50 37.50 15.00 41.25
Sand High 72.50 72.50 75.00 52.50 68.13
Medium 80.00 47.50 100.00 27.50 63.75
Low 82.50 77.50 72.50 22.50 63.75
Phytophthora Loam High 60.00 60.00 85.00 33.79 59.70
Medium 75.00 42.50 92.50 17.50 56.88
Low 75.00 65.00 82.50 5.00 56.88
Sand High 85.00 72.50 87.50 39.91 71.23
Medium 55.00 52.50 82.50 22.50 53.13
Low 77.50 67.50 85.00 7.50 59.38
Low High Nil Loam High 77.50 42.84 72.50 33.43 56.57
Medium 72.50 57.50 95.00 45.00 67.50
Low 62.50 42.50 87.50 10.00 50.63
Sand High 75.00 45.00 65.00 48.45 58.36
Medium 82.50 62.50 90.00 20.00 63.75
Low 75.00 67.50 75.00 22.50 60.00
Phytophthora Loam High 62.50 51.46 82.50 20.72 54.30
Medium 75.00 47.50 90.00 10.00 55.63
Low 55.00 55.00 75.00 12.50 49.38
Sand High 50.00 62.50 75.00 36.71 56.05
Medium 67.50 50.00 95.00 20.00 58.13
Low 65.00 50.00 75.00 10.00 50.00
Low Nil Loam High 65.00 47.50 95.00 40.23 61.93
Medium 70.00 52.50 92.50 25.00 60.00
Low 57.50 20.00 85.00 17.50 45.00
Sand High 45.64 40.17 57.67 47.50 47.75
Medium 72.50 47.50 92.50 27.50 60.00
Low 85.00 75.00 87.50 20.00 66.88
Phytophthora Loam High 76.74 60.00 80.00 26.14 60.72
Medium 70.00 60.00 82.50 17.50 57.50
Low 55.00 47.50 80.00 17.50 50.00
Sand High 50.00 29.57 75.00 30.48 46.26
Medium 70.00 60.00 87.50 15.00 58.13
Low 60.00 62.50 67.50 15.00 51.25
Dry root weight (mg per plant) High High Nil Loam High 51.04 60.43 39.35 74.20 56.26
Medium 40.98 40.00 31.75 38.31 37.76
Low 24.75 30.17 43.20 42.81 35.23
Sand High 67.34 73.97 45.26 82.81 67.35
Medium 60.28 70.11 39.34 55.56 56.32
Low 46.92 45.17 38.08 45.05 43.81
Phytophthora Loam High 34.03 37.46 45.66 28.34 36.37
Medium 27.81 34.03 28.96 27.38 29.55
Low 9.18 13.18 14.35 9.71 11.61
Sand High 73.58 68.11 62.86 9.05 53.40
Medium 36.74 25.20 54.38 7.88 31.05
Low 13.28 14.51 35.41 24.01 21.80
Low Nil Loam High 40.30 41.33 39.95 51.31 43.22
Medium 24.50 31.35 24.37 31.95 28.04
Low 20.88 39.88 26.13 34.33 30.31
Sand High 72.24 70.69 58.25 86.55 71.93
Medium 56.75 43.73 49.30 52.63 50.60
Low 35.55 45.02 52.65 41.94 43.79
Phytophthora Loam High 14.63 14.52 23.65 10.99 15.95
Medium 12.96 17.79 18.77 8.56 14.52
Low 10.58 9.94 13.61 1.91 9.01
Sand High 25.36 23.45 42.22 20.74 27.94
Medium 36.09 34.35 50.31 14.29 33.76
Low 9.95 17.64 24.59 18.88 17.77
Low High Nil Loam High 48.01 65.41 28.21 59.34 50.24
Medium 29.71 19.28 18.93 26.42 23.59
Low 33.66 38.96 28.04 36.01 34.17
Sand High 56.60 55.10 42.63 49.61 50.99
Medium 41.56 29.53 27.36 31.51 32.49
Low 53.32 46.21 45.78 95.09 60.10
Phytophthora Loam High 32.23 20.56 28.31 44.17 31.32
Medium 21.20 15.04 23.36 11.25 17.71
Low 11.23 12.12 10.42 7.14 10.23
Sand High 58.01 40.54 35.12 17.50 37.79
Medium 49.50 43.09 39.07 14.88 36.64
Low 12.50 19.94 34.10 12.57 19.78
Low Nil Loam High 39.94 33.68 26.93 29.97 32.63
Medium 33.04 26.20 24.25 33.25 29.19
Low 15.73 27.34 17.75 17.65 19.62
Sand High 54.90 46.82 40.64 69.13 52.87
Medium 51.19 47.16 40.91 37.81 44.27
Low 29.04 33.59 34.67 40.23 34.38
Phytophthora Loam High 17.55 8.10 23.01 0.00 12.17
Medium 20.63 15.98 22.69 17.50 19.20
Low 9.85 11.84 10.52 15.54 11.94
Sand High 67.67 46.07 25.40 4.26 35.85
Medium 44.54 30.90 37.29 9.38 30.53
Low 27.70 25.42 33.23 13.73 25.02
Dry shoot weight (mg per plant) High High Nil Loam High 183.60 201.40 133.40 278.90 199.33
Medium 114.50 101.40 88.50 68.70 93.28
Low 102.60 85.10 62.90 107.30 89.48
Sand High 313.80 289.20 200.80 467.20 317.75
Medium 178.20 180.30 107.30 129.20 148.75
Low 141.70 116.10 90.00 112.90 115.18
Phytophthora Loam High 90.10 85.20 105.30 10.70 72.83
Medium 50.30 58.60 64.90 37.80 52.90
Low 21.00 29.00 30.60 25.30 26.48
Sand High 181.90 139.40 220.90 22.40 141.15
Medium 58.70 53.30 111.10 7.90 57.75
Low 42.20 46.50 54.50 29.50 43.18
Low Nil Loam High 70.70 68.10 61.70 69.80 67.58
Medium 44.60 48.10 34.40 49.10 44.05
Low 56.70 62.50 40.50 53.10 53.20
Sand High 228.40 164.50 149.40 282.10 206.10
Medium 144.90 97.30 103.70 130.30 119.05
Low 112.60 106.40 118.40 102.30 109.93
Phytophthora Loam High 31.60 21.10 52.10 30.40 33.80
Medium 24.40 35.30 31.70 21.40 28.20
Low 18.50 22.90 19.80 17.30 19.63
Sand High 89.50 65.10 93.70 18.90 66.80
Medium 51.40 55.60 80.50 16.90 51.10
Low 33.20 34.90 40.50 28.60 34.30
Low High Nil Loam High 96.90 151.70 78.30 147.30 118.55
Medium 103.30 78.60 62.30 96.50 85.18
Low 87.80 82.10 77.40 66.50 78.45
Sand High 206.20 138.40 136.90 212.10 173.40
Medium 192.20 87.30 92.00 133.60 251.28
Low 139.90 121.70 100.80 127.80 122.55
Phytophthora Loam High 67.90 44.00 75.70 40.50 57.03
Medium 50.10 35.90 46.20 27.70 39.98
Low 28.20 25.40 34.90 27.30 28.95
Sand High 147.70 93.00 128.40 48.80 104.48
Medium 110.50 83.80 107.30 23.90 81.38
Low 60.40 49.50 56.30 35.60 50.45
Low Nil Loam High 87.50 66.00 49.50 47.80 62.70
Medium 59.30 47.20 50.40 55.00 52.98
Low 37.90 26.80 30.90 23.30 29.73
Sand High 139.30 126.20 92.10 245.30 150.73
Medium 129.90 100.30 84.70 79.60 98.63
Low 70.60 74.80 74.20 77.00 74.15
Phytophthora Loam High 30.80 23.70 45.90 0.00 25.10
Medium 39.90 34.50 35.80 25.80 34.00
Low 30.60 22.40 23.50 15.10 22.90
Sand High 155.40 81.30 54.70 26.80 79.55
Medium 79.60 50.40 85.70 21.40 59.28
Low 69.60 47.00 60.80 31.90 52.33
  • Tap and lateral root disease indices on nil were all 0 and therefore not listed in this Table. ‘Nil’ refers to the treatments without presence of Phytophthora clandestina.
Details are in the caption following the image
Environmental factors affecting percentage emergence of subterranean clover seedlings, and their interactions.

Percentage emergence was also affected by two-way interactions involving moisture and pathogen, moisture and soil type, pathogen and soil type, moisture and temperature, soil type and temperature, moisture and cultivar, pathogen and cultivar, and temperature and cultivar. For example, in the presence of P. clandestina, percentage emergence was significantly higher under high moisture in sand soil and under high temperature with high moisture. In contrast, percentage emergence was lowest at low temperature with either high or low moisture; and low temperature in loam soil significantly reduced emergence. Percentage emergence of cvs Seaton Park and Woogenellup were significantly affected by temperature, with, for example, cv. Seaton Park having greatest germination at intermediate temperature but lowest germination at low temperature. In contrast, cv. Woogenellup germinated best at high temperature but poorest at low temperature.

Percentage emergence was also significantly affected by three-way interactions involving moisture, nutrition and pathogen; nutrition, moisture and cultivar; nutrition, pathogen and cultivar; and nutrition, temperature and cultivar. For example, percentage emergence was lowest in the presence of P. clandestina under low moisture, regardless of nutrition. A major driving factor of three-way interactions was the differences between the subterranean clover cultivars. For example, in terms of interactions between nutrition, temperature and cultivar, cvs Meteora and Seaton Park performed best at intermediate temperature without any impact of nutrition; cv. Riverina performed best under high nutrition and low temperature; and cv. Woogenellup performed best at high temperature and low nutrition conditions.

Percentage emergence was affected by four-way interactions involving moisture, nutrition, pathogen and soil; moisture, pathogen, soil and temperature; nutrition, pathogen, soil and cultivar; nutrition, pathogen, temperature and cultivar; and between nutrition, soil, temperature and cultivar In the presence of the pathogen, across the factors of moisture, temperature, soil type and pathogen, the greatest emergence was for the combination high moisture, high temperature and sand soil. A major driving factor in these four-way interactions was, again, the differences between the subterranean clover cultivars. For example, in the presence of the pathogen, best emergence for cvs Meteora and Seaton Park was under high nutrition with medium temperature; for cv. Riverina it was high nutrition with high temperature; and for cv. Woogenellup it was low nutrition with high temperature.

Tap root disease index

Tap root disease index (%) was significantly affected by single factors of nutrition level, soil type, temperature and subterranean clover cultivar (Tables 2 & 3). In brief, low nutrition, sand soil and high temperature lowered tap root disease index. Cultivar Seaton Park suffered least from tap root disease, followed by cvs Meteora and Riverina, while cv. Woogenellup had the highest levels of tap root disease from P. clandestina. Factors affecting tap root disease index (%) and their interactions are detailed in Figure 2.

Details are in the caption following the image
Environmental factors affecting tap root disease index (%) of subterranean clover seedlings, and their interactions.

Tap root disease was significantly affected by two-way interactions involving soil type and moisture, temperature and nutrition, soil type and temperature, soil type and cultivar, and temperature and cultivar. For example, tap root disease was significantly lower for low moisture with sand soil, for low nutrition with high temperature, for sand soil with high temperature, for sand soil with cv. Seaton Park, and for high temperature with cv. Seaton Park.

Tap root disease was significantly affected by three-way interactions involving moisture, soil and temperature; moisture, nutrition and cultivar; nutrition, soil and cultivar; and soil, temperature and cultivar. Tap root disease was significantly reduced for low moisture, high temperature with sand soil. A major driving factor in these interactions was the differences between the subterranean clover cultivars. For example, tap root disease was significantly reduced for low or high moisture and low nutrition with cvs Meteora, Riverina and Seaton Park (but for high moisture with low nutrition for cv. Woogenellup); for low nutrition and sand soil for all cultivars; for high temperature and sand soil for cvs Meteora and Riverina; for high or low temperature with sand soil for cv. Seaton Park; and for high or low temperature and loam or sand soil for cv. Woogenellup.

Tap root disease was also affected by four-way interactions involving moisture, nutrition, soil and cultivar; moisture, nutrition, temperature and cultivar; and moisture, soil, temperature and cultivar. Again, a major driving factor in these interactions was the differences between the subterranean clover cultivars. For example, tap root disease was reduced for low moisture, low nutrition and sand soil for cvs Meteora, Riverina and Seaton Park; for high moisture, low nutrition and loam soil for cv. Woogenellup; for high moisture, low nutrition and high temperature for cv. Meteora; for high moisture, low nutrition and high or low temperature for cv. Riverina; for high or low moisture, low nutrition and high or low temperature for cv. Seaton Park; for high moisture, low nutrition and high temperature for cv. Woogenellup; for high or low moisture, sand soil and high temperature for cv. Meteora; for high moisture, sand soil and high temperature for cv. Riverina; for high moisture or low moisture, sand soil and high temperature for cv. Seaton Park; and for high moisture, loam soil and high temperature for cv. Woogenellup.

Lateral root disease index

Lateral root disease index (%) was significantly affected by single factors of moisture, nutrition, soil type, temperature and cultivar. In brief, lateral root disease index was reduced at high moisture, by low nutrition, in sand soil, by high temperature and by using cv. Seaton Park (Tables 2 & 3). Factors affecting lateral root disease index (%) and their interactions are detailed in Figure 3.

Details are in the caption following the image
Environmental factors affecting lateral root disease index (%) of subterranean clover seedlings, and their interactions.

Lateral root disease index was significantly affected by two-way interactions involving moisture and soil; nutrition and temperature; soil and temperature; moisture and cultivar; soil and cultivar; and by temperature with cultivar. For example, lateral root disease index was reduced for low moisture with sand soil; for high temperature with low nutrition; for high temperature with sand soil; for high moisture with cv. Seaton Park; for sand soil with cv. Seaton Park; and for high or low temperature with cv. Seaton Park.

Lateral root disease index was significantly affected by three-way interactions involving moisture, nutrition and soil; between moisture, nutrition and cultivar; between nutrition, soil and cultivar; between moisture, temperature and cultivar; and between nutrition, temperature and cultivar. For example, lateral root disease index was reduced for low moisture, low or high nutrition and sand soil; for high moisture, low nutrition and cv. Seaton Park; for high nutrition, sand soil and cv. Seaton Park; for high moisture, high or low temperature and cv. Seaton Park; and for low nutrition, high temperature and cv. Seaton Park.

Lateral root disease index was also significantly affected by four-way interactions involving moisture, nutrition, soil and temperature; between moisture, nutrition, soil and cultivar; between moisture, nutrition, temperature and cultivar; and between moisture, soil, temperature and cultivar. For example, lateral root disease index was significantly reduced for high moisture, low nutrition and high temperature in loam soil; for low moisture, low nutrition and sand soil for cvs Meteora, Seaton Park and Riverina; for high moisture, low nutrition and loam soil for cv. Woogenellup; for high moisture, high nutrition and high temperature for cvs Meteora and Riverina; for high moisture, low nutrition and high temperature for cv. Seaton Park; for high moisture, low nutrition and high temperature for cv. Woogenellup; for high or low moisture, sand soil and high temperature for cv. Meteora; for low moisture, sand soil and high or medium temperature for cv. Riverina; for low moisture, sand soil and all three temperatures for cv. Seaton Park; and for high moisture, loam soil and high temperature for cv. Woogenellup. As with tap root disease, a major driving factor in these interactions was the differences between the subterranean clover cultivars.

Nodulation index

Although overall nodulation levels were generally very low (range for percentage nodulation indices of 0–25.6; data not shown), nodulation was significantly affected by single factors of nutrition, pathogen, soil type, temperature and cultivar (Table 2). In brief, the nodulation index was higher when soil nutrition was low, when P. clandestina was present, when sand soil was used, when temperature was high, and when cv. Seaton Park was the cultivar used. Factors affecting nodulation index (%) and their interactions are detailed in Figure 4.

Details are in the caption following the image
Environmental factors affecting nodulation index (%) of subterranean clover seedlings, and their interactions.

Nodulation was significantly affected by two-way interactions involving nutrition and pathogen, nutrition and soil, pathogen and soil, nutrition and temperature, pathogen and temperature, soil and temperature, moisture and cultivar, nutrition and cultivar, pathogen and cultivar, soil and cultivar, and temperature and cultivar (data not shown). For example, nodulation increased for low nutrition and the presence of P. clandestina, for low nutrition and sand soil, for presence of P. clandestina and sand soil, for high temperature and low nutrition, for high temperature and the presence of P. clandestina, for high temperature and sand soil, for high moisture and cv. Seaton Park, for low moisture and cvs Meteora or Riverina, for low nutrition with all tested cultivars, for the presence of P. clandestina and all cultivars, for sand soil and all cultivars, and for high temperature and all cultivars.

Nodulation was also significantly affected by three- and four-way interactions involving the different factors (data not shown). For example, nodulation index was increased by the three-way interactions involving low nutrition, the presence of P. clandestina and sand soil; for low nutrition, the presence of P. clandestina and high temperature; for low nutrition, sand soil and high temperature; for sand soil, high temperature and the presence of P. clandestina; for low moisture, the presence of P. clandestina and cvs Meteora, Riverina (or for cv. Woogenellup with high moisture), and for low moisture with the presence of P. clandestina on cv. Seaton Park; for high moisture, sand soil and cv. Seaton Park; for sand soil, the presence of P. clandestina and all cultivars; for low moisture, high temperature and cvs Meteora or Riverina (or high moisture, high temperature and cv. Seaton Park); for low nutrition, high temperature and cvs Meteora, Riverina and Seaton Park; for high temperature, the presence of P. clandestina and all cultivars; and for sand soil, high temperature and all cultivars. Similarly, nodulation increased in situations involving four-way interactions for low nutrition, the presence of P. clandestina, sand soil and high temperature; in particular, it increased for cvs Meteora, Riverina and Seaton Park in situations involving the presence of P. clandestina, low nutrition, and sand soil.

Dry root weight

Dry root weight (mg per plant) was significantly affected by the single factors of moisture, nutrition, pathogen, soil, temperature and cultivar (Tables 2 & 3). In brief, dry root weight was increased by high moisture, by high nutrition, by the absence of P. clandestina, by sand soil, by high temperature and by using cvs Meteora or Riverina. Factors affecting dry root weight and their interactions are detailed in Figure 5.

Details are in the caption following the image
Environmental factors affecting dry root weight (mg per plant) of subterranean clover seedlings, and their interactions.

Dry root weight was significantly affected by two-way interactions involving moisture and pathogen, nutrition and soil, moisture and temperature, nutrition and temperature, pathogen and temperature, moisture and cultivar, pathogen and cultivar, soil and cultivar, and temperature and cultivar. For example, dry root weight was increased for high moisture and the absence of P. clandestina, for high nutrition and sand soil, for high moisture and high or medium temperature, for high temperature and high nutrition, for high temperature and the absence of P. clandestina, for high moisture and cvs Riverina or Seaton Park, for the absence of P. clandestina and all cultivars and especially cv. Woogenellup, for sand soil and all cultivars and especially Meteora, Riverina and Seaton Park, and for high temperature and cvs Meteora or Riverina.

Dry root weight was significantly affected by three-way interactions involving moisture, nutrition and pathogen; for moisture, nutrition and temperature; for nutrition, pathogen and temperature; for nutrition, soil and temperature; for pathogen, soil and cultivar; for pathogen, temperature and cultivar; and for soil, temperature and cultivar. For example, dry root weight increased for high moisture, high nutrition and the absence of P. clandestina; for high moisture, high nutrition and high temperature; for high nutrition, the absence of P. clandestina and high temperature; for high nutrition, sand soil and high temperature; for the absence of P. clandestina, sand soil and all tested cultivars and especially cv. Woogenellup; for the absence of P. clandestina, high temperature and cvs Woogenellup or Meteora or Riverina; and for sand soil, high temperature and cvs Meteora or Riverina.

Dry root weight was also significantly affected by four-way interactions involving moisture, nutrition, pathogen and temperature; between moisture, pathogen, soil and cultivar; and between nutrition, soil, temperature and cultivar. For example, dry root weight increased for high moisture, high nutrition, the absence of P. clandestina and high temperature; for high moisture, the absence of P. clandestina, sand soil and cvs Meteora or Riverina or Woogenellup; for high nutrition, sand soil, high temperature and cvs Meteora or Riverina; and for the five-way interaction of high moisture, low nutrition, the absence of P. clandestina, sand soil and high temperature.

Dry shoot weight

Dry shoot weight (mg per plant) was significantly affected by the single factors of nutrition, pathogen, soil, temperature and cultivar (Tables 2 & 3). In brief, dry shoot weight was increased by high nutrition, by the absence of P. clandestina, by sand soil, by high temperature, and by using cv. Meteora. Factors effecting dry shoot weight and their interactions are detailed in Figure 6.

Details are in the caption following the image
Environmental factors affecting dry shoot weight (mg per plant) of subterranean clover seedlings, and their interactions.

Dry shoot weight was significantly affected by two-way interactions involving nutrition and pathogen, for pathogen and soil, for moisture and temperature, between nutrition and temperature, for pathogen and temperature, for soil and temperature, and for pathogen and cultivar. For example, dry shoot weight increased under the conditions of high nutrition and the absence of P. clandestina, sand soil and the absence of P. clandestina, under high moisture and high temperature, high nutrition and high temperature, high temperature and the absence of P. clandestina, sand soil and high temperature, and the absence of P. clandestina and use of cv. Woogenellup.

Dry shoot weight was significantly affected by three-way interactions involving moisture, nutrition and temperature; and for pathogen, temperature and cultivar. For example, dry shoot weight increased for high nutrition, high temperature and high moisture; for absence of P. clandestina, with high temperature and cv. Woogenellup; and for absence of P. clandestina with medium temperature and Meteora.

Relationships between important variables

There was a significant and strong positive relationship between tap root disease index (%) and lateral root disease index (%) (= 1.0864x − 5.8244; < 0.001, = 94; R2 = 0.92). There was a significant negative relationship between tap root disease index (%) and dry root weight (mg per plant) (= −0.3915x + 43.196; < 0.001, = 94; R2 = 0.34). There was a significant negative relationship between lateral root disease index (%) and dry root weight (mg per plant) (= −0.3349x + 39.73; < 0.001, = 94; R2 = 0.33). There was a significant negative relationship between tap root disease index (%) and dry shoot weight (mg per plant) (= −0.8204x + 85.916; < 0.001, = 94; R2 = 0.29). There was a significant negative relationship between lateral root disease index (%) and dry shoot weight (mg per plant) (= −0.7324x + 80.646; < 0.001, = 94; R2 = 0.30). There was a significant and strong positive relationship between dry root weight (mg per plant) and dry shoot weight (mg per plant) (= 2.194x − 1.4134; < 0.001, = 94; R2 = 0.78).

Discussion

These are the first studies undertaken to comprehensively define the importance of environmental conditions occurring across southern Australian subterranean clover forage pastures on the severity of phytophthora damping-off and root rot and on plant productivity. Environmental factors significantly affected severity of pre-emergence damping-off, root disease and root and shoot productivity, high moisture, high or medium temperature, high nutrition, sandy rather than loamy soil and subterranean clover cultivars with better resistance to P. clandestina all contributed towards less damping-off and root disease, and to greater subterranean clover productivity. There were significant interactions between temperature, moisture soil type and nutrition, particularly in relation to pre-emergence damping-off and tap and lateral root disease.

In the presence of P. clandestina, the level of tap root disease was significantly affected by temperature and cultivar, while the level of lateral root disease was significantly affected by both moisture and temperature. There have been a number of historical attempts to relate some environmental factors to the severity of root diseases of subterranean clover ecosystems in southern Australia. For example, MacNish et al. (1976) found that years of heavier and more frequent rain after the opening seasonal rains led to more severe root disease severity than years of lower rainfall and moisture. While it is widely accepted that oomycete pathogens like P. clandestina are strongly favoured by wet soil conditions, it was surprising, in the current study, that the higher of the two moisture levels (100% WHC) contributed towards less root disease and less damping-off than 50% WHC. However, this is similar to the findings of Wong et al. (1984) for P. irregulare where most severe root rotting occurred at 65% WHC, with less at 45% WHC, and, surprisingly, least root disease under flooding conditions. However, in contrast, other studies suggest that, generally, P. clandestina and another oomycete common across southern Australia, A. trifolii, are favoured by high moisture conditions (e.g. Wong et al., 1986b,c; O'Rourke et al., 2010). Wong et al. (1986c) found that the most severe root disease from P. clandestina occurred at a soil temperature of 10 °C, followed by 15 and 20 °C, coinciding with late autumn/early winter temperature conditions of the Mediterranean ecosystems of southern Australia. Further, Wong et al. (1986c,e) showed that there was a marked reduction in the growth of P. clandestina with increasing water stress across the range of temperatures, as could be expected in the field, suggesting that even short periods of low soil moisture could be more important than the variations in temperature expected to occur across southern Australia. However, in the current study it was clear that the lower of the two moisture levels (viz. 50% WHC) was adequate for P. clandestina to cause severe damping-off and root disease.

In the presence of P. clandestina the level of both tap and lateral root disease was significantly affected by nutrition. Similarly, O'Rourke et al. (2012) highlighted the potential of incorporation of nutrient amendments into an integrated and more sustainable approach to better manage root disease and to increase productivity of subterranean clover, particularly where soils are inherently deficient in one or more nutrients. O'Rourke et al. (2012) found that application of a complete nutrient solution to field soils decreased the severity of tap and lateral root disease by approximately 45% and 32%, respectively; and that even amendment with either K or N alone could reduce the severity of tap root disease by >30%. Current field experiments across southern Australia have also highlighted significant increases of up to 1.5-fold in productivity of subterranean clover pastures severely affected by root disease from complete nutrient application (M. P. You, unpublished data). Thus, improvement of nutrition offers significant potential for mitigation of the disease severity and impact caused by P. clandestina.

In the presence of P. clandestina, seedling emergence (i.e. level of pre-emergence damping-off) was significantly affected by moisture, soil type, temperature and cultivar. Wong et al. (1986c) similarly demonstrated such effects with P. clandestina; they examined pre- and post-emergence damping-off in subterranean clover under a range of soil temperature (10–30 °C) and moisture conditions (65% and 100% WHC and flooding), finding the greatest reductions in seedling survival occurring in saturated and flooded soil. In the present study, in the presence of P. clandestina, pre-emergence damping-off was less in sand soil than loam soil; clearly, soil type can be an important determining factor in relation to severity of disease from P. clandestina. Despite such an effect of soil type, P. clandestina remains a serious pathogen throughout a wide variety of different soil types across southern Australia, from coarse sand to heavy clay soils (authors’ unpublished data). In relation to strong interactions between nutrition, pathogen, soil type and cultivar, it is noteworthy that cv. Seaton Park showed the least pre-emergence damping-off, followed by cvs Meteora, Riverina and Woogenellup, in all combinations; cv. Seaton Park has also been observed to perform the best of these cultivars in areas where soilborne disease from P. clandestina is prevalent (M. P. You, unpublished data). Temperature and moisture have long been recognized as major drivers for plant seed germination and emergence. Furthermore, interactions among climatic conditions, soil, seeds and seedling characteristics in relation to seedling emergence are complex under field conditions (Whalley et al., 1999; Vleeshouwers & Kropff, 2000), even without presence of one or more serious pathogens. The environmental conditions directly surrounding a seed determine germination success and subsequent seedling emergence and establishment (Harper, 1977); the presence of soilborne pathogens like P. clandestina make germination and establishment of seeds more challenging. As found in the current study for soils infested with P. clandestina, soil temperature and soil water potential are well recognized major driving factors for seed germination and seedling emergence (Gummerson, 1986; Finch-Savage & Phelps, 1993; Dahal & Bradford, 1994).

It has long been observed that the above-ground symptoms of root rots in subterranean clover vary in different localities (Barbetti et al., 2007), and while the aboveground symptoms can be a consequence of different complexities of the different combinations of soilborne pathogens involved (Barbetti et al., 2007), it has been believed that this could be due to environmental differences (Barbetti & MacNish, 1983), cultivar (Barbetti et al., 1986b; Barbetti, 1989) and nutritional influences (M. J. Barbetti, unpublished data). For example, typical symptoms can include stunted yellow-green, or yellow-red, or red-purple plants (Burgess et al., 1973; Barbetti & MacNish, 1983; Clarke, 1983) either scattered among apparently healthy plants or in distinct patches (Barbetti & MacNish, 1983; Clarke, 1983). However, it still remains to be proven that such symptom variations in the field are due to the influence of environmental factors on soilborne pathogens rather than variations in the composition of different pathogen complexes.

It is noteworthy that the tap and lateral root disease were so strongly and positively correlated, at least for the four cultivars used in the current study. This suggests that, at least for some cultivars, it is possible that tap and lateral root disease resistance(s) may be controlled by similar gene(s) or host resistance mechanism(s). If so, then this opens scope for screening and breeding for tap and lateral root rot disease resistances simultaneously. However, first, any similarity in genetic control of host resistance would have to be confirmed across the subterranean clover genotypes to be used in any breeding programme aiming to develop more disease-resistant cultivars. Both tap and lateral root disease indices were strongly negatively correlated with dry root or shoot weights, which, again, highlights the critical requirement to maintain healthy tap or lateral roots if subterranean clover productivity is to be improved over current low levels. It has already been well established in other studies that plants with more severe root disease have less root or shoot productivity (e.g. Barbetti et al., 1986a,b; Wong et al., 1986a) and the current study further confirms this conclusion. The close relationship between root and shoot weights confirms, as expected, the critical need for healthy root systems if shoots are to be productive, as found in earlier studies (e.g. Barbetti et al., 1986a,b).

Cultivar of subterranean clover significantly affected the level of both tap and lateral root disease in the presence of P. clandestina, as observed for pre-emergence damping-off (discussed above). Cultivar host resistance was critical for reducing root disease severity and increasing productivity, even when favourable environmental conditions for severe disease occurred. For example, in the presence of P. clandestina, cv. Seaton Park performed best under a high temperature, high nutrition and high moisture combination, but showed poor productivity under conditions of low nutrition or lower temperature. A major driving factor in these interactions was the differences between the subterranean clover cultivars. Cultivar Seaton Park is the only cultivar of the four chosen for this study that has resistance to race 173 (You et al., 2005a,b). In contrast to cv. Seaton Park, less resistant cvs Riverina and Meteora had less disease and greater productivity under low moisture conditions, less favourable for P. clandestina. This was expected as You et al. (2005b) demonstrated that cv. Meteora was moderately resistant to race 173 used in the current study. This highlights the importance and advantages of breeding disease-resistant cultivars that cannot only overcome disease problems per se but also unfavourable environmental conditions. Cultivar Woogenellup is known to be highly susceptible to P. clandestina (Barbetti, 1989; You et al., 2005a,b). However, a major challenge in managing this pathogen remains: the pathogenic specialization of P. clandestina changes in response to the cultivar or spectrum of cultivars used in a given geographic region (You et al., 2006; Nichols et al., 2014).

The extent of Rhizobium nodulation of the clover was significantly affected by nutrition, pathogen, soil type, temperature and cultivar; with more nodulation when soil nutrition was low, when P. clandestina was present, when sandy soil was used, when temperature was high, and when cv. Seaton Park was the cultivar used. The stimulation of nodulation by the presence of P. clandestina was unexpected, as reductions in root rot severity have historically been obtained from inoculating seed with rhizobia (M. J. Barbetti, unpublished data); in addition, in glasshouse studies, a strain of Rhizobium trifolii was found to significantly reduce root rot caused by another soilborne pathogen, Fusarium avenaceum (Wong, 1986). This finding in the current study cannot, at present, be explained, but this effect has been observed in previous investigations (M. P. You, unpublished data).

Soilborne pathogens predominate across much of southern Australia as the Mediterranean-type climate fosters survival of these pathogens on infested residues through dry summer periods (Wong et al., 1986e). However, as global warming leads to climate change, significant changes in the relative importance of pathogens, including soilborne pathogens, are likely to occur across southern Australia (Chakraborty et al., 1998; Barbetti et al., 2012; Jones & Barbetti, 2012). Climate changes, such as an extension of effective summer rain further southwards, particularly in eastern Australia, will lead to increased rates of breakdown of infested soilborne residues, especially as average winter rainfall is falling across southern Australia (Barbetti et al., 2012). Further investigations may help to explain some of the changes in severity of disease caused by P. clandestina or the relative distributions and incidence of individual soilborne pathogens in relation to climate change. This has already been noted for some other pathogens of subterranean clover; for example, in Western Australia, there has been a distinct contraction southwards of the subterranean clover area affected by the northern anthracnose pathogen, Kabatiella caulivora, as the climate in the southwest of that state becomes increasingly drier and warmer (Jones & Barbetti, 2012). Such environmental effects may also explain the variability and differences between areas across southern Australia affected by root rot in relation to foliar symptoms of plants and variation in root disease severity; they may even explain, to some extent, the variation in performance of host resistance that occurs. While it is clear that fluctuating temperature and moisture conditions, so common under the southern Australian annual subterranean clover pastures, have a significant impact on the severity of disease epidemics and the expression of host resistances, it is important to realize that the temperature thresholds vary with different pathogens and/or their combinations (Barbetti, 1984). In addition, temperature and moisture interact with soil type, nutrition and even cultivar (current study), further complicating any attempts to associate specific above-ground plant symptoms with particular environmental conditions or situations.

Across southern Australia, producers usually face critical feed shortages during autumn and winter that coincide with severe attack by soilborne pathogens such as P. clandestina. This markedly decreases the autumn–winter biomass production in regenerating stands. While the extent of disease and even the disease symptoms remain highly variable across southern Australia, this is the first study to attempt to highlight the complexity of interactions driving such variability. In particular, while there is a strong relationship between early-season subterranean clover production and seedling density (Donald, 1951), it has remained unknown until now that soilborne pathogens such as P. clandestina cause such fluctuating, but often severe, pre-emergence damping-off and/or decreases in seed yield, resulting in subsequent failure of regenerating clover forages. Currently, severely affected forages require application of a combination of expensive cultural management techniques (e.g. cultivation) and reseeding with more disease resistant cultivars if they are available (Barbetti et al., 2006, 2007). Improved host resistance offers the best long-term and cost-effective way to curtail losses where severe phytophthora root rot and pre-emergence damping-off in subterranean clover occurs, particularly as the disease severity is clearly driven by moisture, temperature, nutrition, soil type and cultivar. The current study demonstrates how variations in environmental factors such as soil type, nutrition, moisture and temperature, and also cultivar, individually and interactively, can have profound effects on the expression and severity of phytophthora pre-emergence damping-off and root disease and the consequent level of productivity of subterranean clover forage.

Acknowledgements

The authors thank the Meat and Livestock Australia for funding this research via project ‘B PSP 0005 – Managing Soil-borne Root Disease in Sub-clover Pastures’. The authors have no conflict of interest to declare.