Introduction

Kappaphycus alvarezii (Doty)Doty ex P.C. Silva, a raw material for the extraction of the phycocolloid kappa carrageenan, is considered an important cultivated, commercial marine commodity in the tropics, particularly in the Philippines, Indonesia and Malaysia (see Bixler and Porse, 2010 for a review) However, there are many different varieties of K. alvarezii, two of which are K. alvarezii var. tungawan and K. alvarezii var. giant tambalang. K. alvarezii var. tungawan is characterized by its thin, rather slender, cylindrical thallus with sharp attenuating tips while K. alvarezii var. giant tambalang is characterized by long and wide cylindrical thallus (1.0–1.5 cm diameter) and sparse branches with sharp pointed apices.

Commercial farming of Kappaphycus is very dependent on the local cultivation conditions, management practices and any outbreak of seaweed disease or pest species. The two major disease outbreaks encountered in seaweed farms are “ice-ice disease” and infestation of epiphytes (filamentous red algae) which cause thallus degradation (Ask and Azanza 2002).

“Ice-ice” disease of Kappaphycus is reportedly caused by microbes identified as the Vibrio–Aeromonas complex and Cytophaga–Flavobacterium complex, as triggered by stress due to extreme environmental conditions such as low salinity or low light intensity (Largo et al. 1995a). Meanwhile, epiphyte infection in Kappaphycus farms is primarily caused by red filamentous macroalgae, which include Neosiphonia spp. (Hurtado et al. 2006; Vairappan et al. 2008). Neosiphonia spp. outbreaks are associated with significant changes in seawater temperature and salinity (Vairappan 2006). Neosiphonia spp. infestation initially appears with the appearance of tiny black spots on the cuticle of carrageenophyte host, followed by the emergence of red hair-like filaments; and finally by the presence of small elevated pores or “goosebumps” (Vairappan et al. 2008) on the host’s surface that are the sites of penetration from the cortical to the medullary layers of the host plant. Neosiphonia spp. infestation also caused the seaweeds to weaken; making them susceptible to secondary bacterial infection and subsequent breaking of the thallus into small pieces that fell from the production lines and as such are lost biomass. Epiphyte outbreaks in Malaysia and the Philippines appeared to be seasonal (February–March and September–November) and resulted in a significant reduction of commercial biomass production and an associated decline in carrageenan quality characteristics (Vairappan et al. 2008).

Ascophyllum nodosum is a marine, brown alga that grows abundantly on the rocky intertidal shores of Atlantic Canada and northern Europe (Ugarte et al. 2006). Commercial extracts of this seaweed species, either in the form of liquid products or soluble powder, have been used as a source of fertilizer and soil amendment or conditioner in a number of agricultural production systems (see Cragie 2010 for an extensive review). Studies using commercial extracts of the brown alga A. nodosum as a soil drench and spray have shown that they can improve plant growth rates, enhance resistance to abiotic stress factors (i.e., frost and salinity), and reduce the impacts of biotic stresses such as fungal diseases and insect infestation. Alleviation of such stress factors consequently increases crop yields, as well as improving the overall quality and value of the products to which the extract is applied (Blunden et al. 1996; Leach et al. 1999). Extracts from A. nodosum, both as soluble powder and as liquid extract, promoted root–tip elongation. and shoot growth in the model plant Arabidopsis thaliana (Rayorath et al. 2007) as well as in barley (Rayorath et al. 2008). Several studies also showed that A. nodosum extracts significantly reduced the effects of both bacterial and fungal-induced disease in grapes (Lizzy et al. 1998), carrot (Jayaraj et al. 2008) and A. thaliana (Rayorath et al. 2007).

Recently, the effect of the commercial A. nodosum extract was also tested in K. alvarezii in vitro. The results showed that the extract was effective in improving micro-propagation of several different varieties of Kappaphycus using tissue culture techniques (Hurtado et al. 2009) and in improving the growth of K. alvarezii in the Philippines and reducing the presence of some epiphytes such as Ulva and Cladophora in Brazil (Loureiro et al. 2009).

The present study was conducted to determine the efficacy of Acadian Marine Plant Extract Powder (AMPEP) on the growth and occurrence of Neosiphonia infestations on two varieties of K. alvarezii, suspended at different depths in the sea, in the Philippines, a major commercial center for production of this important carrageenophyte.

Materials and methods

The study was conducted at the Igang Marine Station of the Southeast Asian Fisheries Development Center Aquaculture Department, Igang Nueva Valencia, Guimaras (10°02′N and 122°02′E).

Vigorous and Neosiphonia-free propagules of K. alvarezii (Tungawan (TUNG) and giant tambalang (GTAM)) were cultivated at 0, 50, 100, and 150 cm below the water surface (Hurtado et al. 2008). Approximately 100 g of test sample propagules were tied individually to one end of a 10-mm diameter polyethylene rope; the other end was tied to a bamboo pole placed on top of a floating net cage. The length of cultivation line was adjusted in order to provide the desired depths. Prior to out-planting, each seedling was soaked in 0.1 g L−1 of AMPEP—a commercial extract from A. nodosum—for 30 min (Hurtado and Critchley, unpublished data). Five replicates for each depth level were prepared with a corresponding number of controls (e.g., no dipping in AMPEP).

At the end of each 45-day culture period, the daily growth rate and percent cover of Neosiphonia infestation were evaluated from April to November. After a 45-day period, healthy plants were pruned to produce new 100-g test seedlings, which were then used as new propagules for each succeeding culture period.

The average daily growth rate (DGR = % day−1) was expressed as the percentage increase in fresh weight per day for each replicate which was calculated according to the formula:

$$ {\hbox{DGR}} = \left[ {{ \ln }\left( {{\hbox{ Final weight}}/{\hbox{initial weight }}} \right)/{\hbox{duration of culture }}} \right] \times {1}00 $$

The percent occurrences of “goosebumps” and “Neosiphonia sp.” were estimated as percent cover using a scale of 1–10 (1 = 10%, 2 = 20%…10 = 100%). Numbers 1–10 were assigned visually from the apical, mid, and basal sections of the Kappaphycus thalli. The average of the three sections was converted to percent in the laboratory for ease of computation.

Environmental parameters, at each depth level, were measured weekly for 24 h at 3-h intervals. Seawater salinity, temperature, and turbidity were measured using a multi-probe water quality checker (YSI 650 MDS). Irradiance was measured with a LI-250 light meter.

Results were analyzed statistically (two-way ANOVA) at the 5% level of significance using GraphPad Prism Ver. 5.1 software. Correlation analyses (r) between percent occurrence of Neosiphonia sp. and some environmental factors were determined at the 5% level of significance.

Results

After 45 days of culture, both K. alvarezii varieties treated with AMPEP had significantly higher growth rates (e.g., 1.3–4.1% day−1) than the control (e.g., 0.9–3.0% day−1). A decreasing growth rate of the two varieties was also observed with increasing depth of culture. AMPEP dipped seaweeds grown at the water surface had the highest growth rates (viz. 4.1%, TUNG; 3.1%, GTAM); while the control at 150 cm had the lowest growth rates (1.2%, TUNG; 0.9%, GTAM; Table 1).

Table 1 Average (±SD) daily growth rate and percent occurrence of Neosiphonia sp. on K. alvarezii var. giant tambalang, and var. tungawan with and without AMPEP dipping grown at four depths from April to November (n = 5)
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The percent occurrence of Neosiphonia sp. infections on both varieties dipped in AMPEP (6–36% at all depths) were significantly lower than the control (7–69% at all depths). The percent occurrence of Neosiphonia sp decreased as the cultivation depth increased (Fig. 1a and b). The first occurrence of Neosiphonia sp infection was observed 21 days after culture began from June to November. The peak of the Neosiphonia sp. outbreak was recorded in July.

Fig. 1
figure 1

a Average percent occurrence of Neosiphonia sp. on K. alvarezii var. giant tambalang grown at different depths from April to November. b Average percent occurrence of Neosiphonia sp. on K. alvarezii var. tungawan grown at different depths from April to November

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The average temperature, salinity, and irradiance readings from April to November ranged from 28.9 to 29.7°C, 31.7 to 33.5 ppt, and 194 to 716 μmol photons m−2 s−1, respectively (see Table 2). The lowest readings of temperature, salinity, and irradiance were taken between July and August at all depths studied. The emergence of epiphytes in June–July coincided with an observed decrease in salinity from 33.3 to 31.7 ppt.

Table 2 Average irradiance, salinity, temperature, and turbidity at different depths from April to November
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The correlation between physical environmental factors and the occurrence of Neosiphonia sp. infestations at different depths, on both varieties of K. alvarezii, is shown in Table 3. In both varieties, a strong and positive correlation was found between irradiance and percent occurrence of Neosiphonia (r = 0.94–1.00); a negative but strong correlation between salinity and percent occurrence of Neosiphonia (r = −0.95 to −0.99); a positive but weak, correlation between temperature and percent occurrence (r = 0.21–0. 47); and a negative and weak correlation between turbidity and percent occurrence of Neosiphonia (r = −0.03 to −0.25).

Table 3 Correlation coefficient between percent occurrence of Neosiphonia sp. and some environmental parameters in two varieties of K. alvarezii
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Discussion

The recent review of Cragie (2010) gave an extensive account of the use of seaweed extracts with agricultural crop production. However, there are few and limited reports of the use of seaweed extract as a growth promoter in commercially grown seaweeds. Loureiro et al. (2009) reported on the in vitro growth rate of three variants of K. alvarezii treated with extract from A. nodosum to have higher growths (e.g., 5.9% day−1) when compared to their control (2.4% day−1). The lower growth rates (with AMPEP, 1.3–4.1% day−1 and control 0.9–3.0% day−1) obtained in the present study are most likely attributable to the concentration of AMPEP and the culture methods applied. The work of Loureiro et al. (2009) was conducted under laboratory conditions while the present study was conducted in the field, exposed to the varied and uncontrolled elements. In another study, undertaken in South Africa on an extract made from the kelp Ecklonia maxima, the effects of: turbot effluents + fertilizer, turbot effluents + fertilizer + Kelpak, and turbot effluents + Kelpak on the growth of Ulva lactuca were not significantly different from one another, although growth rates were increased (viz. 5.1–6.6% day−1); there was a significant difference from the untreated Ulva (Robertson-Anderson et al. 2006). These studies provide an indication that the green alga Ulva requires additional nutrients for improved growth, which was similar to the results obtained in from the two varieties of K. alvarezii in the present study. Unfortunately, Robertson-Anderson et al. (2006) did not include the effects of Kelpak applied individually on the growth of Ulva to assess if it could be used as a stand-alone growth enhancer for commercial seaweed cultivation.

Though earlier reports on the cultivation of K. alvarezii showed higher growth rates in the Philippines than the present study (e.g., 5.7% day−1, Lim and Porse 1981; 4.4–8.9% day−1, Dawes et al. 1994); Brazil (6.5–10.7% day−1, de Paula et al. 2002); Vietnam (7–11% day−1, Ohno et al. 1996), the additional reduction of the occurrence of Neosiphonia infestation using AMPEP was very encouraging and provides a potential management tool. Neosiphonia infestation was first reported in the Philippines in Calaguas Island, Camarines Norte (Critchley et al. 2004) and subsequently became a problem in other parts of the Philippines (Hurtado 2004). The perennial occurrence of Neosiphonia infestation in the Philippines since early 2000 led to the reduced availability of good quality cultivars and seedlings for commercial farming (Critchley et al. 2004; Hurtado and Critchley 2006) and consequently lower productivity. Vairappan (2006) and Vairappan et al. (2008) reported on the spread of the malady to Malaysia.

The use of commercial, Ascophyllum extract powder as a plant growth promoter was tested in A. thaliana as a model plant (Rayorath et al. 2007) and similar results were observed to the present study. Young, lateral shoots started to appear on the Kappaphycus varieties in the present study after 10–15 days of culture, these later developed into branches as the duration of cultivation progressed. Furthermore, Rayorath et al. (2008) showed that the organic components of the commercial A. nodosum extract they used induced amylase activity which was independent of GA3 and which might act in concert with GA-dependent amylase production leading to enhanced germination and seedling vigor in barley. The observations of the authors in barley was also apparent in both varieties of K. alvarezii which were dipped in AMPEP in the present study, where a smooth thallus surface and brittle branches were observed, indicative of a healthy and vigorous plant.

Several studies have reported on the efficacy of commercial extracts of A. nodosum on land-based crops for improving growth, yield and quality, pest and disease resistance, and environmental stress tolerance (Rayorath et al. 2007; 2008; Jayaraj et al. 2008; 2010). The significant reduction in percent occurrence of Neosiphonia infestation in both varieties of K. alvarezii treated with AMPEP showed parallel results with the following land-based crops which were also treated with various forms of A. nodosum extract: significant reduction in black bean aphid (Aphis fabae) infestations on broad bean (Stephenson 1966); reduction in fecundity of the root-knot nematode (Meloidogyne javanica) on Arabidopsis seedlings (Wu et al. 1998); a significantly reduced severity of Alternaria radicina in carrots which was probably caused by the induction of defense genes or proteins (see Jayaraj et al. 2008); enhanced disease resistance to Alternaria cucumerinum through induction of defense genes or enzymes in cucumber (Jayaraj et al. 2010); reduction in dollar spot disease (Sclerotinia homoeocarpa) on bent grass (Zhang et al. 2003); and reduced root damage from nematode (Meloidogyne incognita) predation in tomatoes using an extract from Ecklonia maxima (Featonby-Smith and van Staden 1983).

The decrease in percent occurrence of Neosiphonia sp. infection was observed with increasing depth of cultivation. Seaweeds grown with prior dipping in AMPEP at the surface of the water had the highest growth rate, those without AMPEP dipping had the highest percent occurrence of Neosiphonia infection. These results also compared well to a study by Hurtado et al. (2008), using K. alvarezii var. sacol as test samples. They reported that K. alvarezii var. sacol had the highest growth rate at depths of 50–100 cm.

The studies by Hurtado et al. (2006) and Vairappan (2006) reported that the emergence of an epiphytic outbreak is a complex problem with the extent of the outbreak being dependent on the quality of the cultivated strain, abiotic parameters of the culture site, management practices, and seasonal weather fluctuations.

In the present study, the lowest surface, seawater temperature (e.g., 28.9°C) and salinity (31.7–32.1 ppt) readings were recorded from July to September; these coincided with the rainy season and also the highest percent occurrence of Neosiphonia (i.e., GTAM, 55–85%; TUNG, 63–85%). The highest percent occurrence of Neosiphonia infection was observed in July, thus corresponding to the period of lowest salinity and irradiance. Slightly higher surface seawater temperatures and salinities were recorded from April–May, the time at which the higher growth rates of Kappaphycus with 0% occurrence of Neosiphonia on both TUNG and GTAM were recorded in the cultivation area. These results further confirmed the study of Vairappan (2006), in which a correlation between the fluctuations in the abiotic factors and the emergence of Neosiphonia epiphytes were reported for Malaysia. Significant changes in abiotic factors have also been reported to trigger bacterial diseases in Japanese kelp, Eucheuma denticulatum and K. alvarezii (Glenn and Doty, 1990; Largo et al. 1995a, b).

The positive and strong correlations between irradiance and percent occurrence of Neosiphonia indicated a linear function of the two variables. Thus, 94–100% of the variation in the percent occurrence of Neosiphonia could be accounted for by the linear function of the variation in the mean of the irradiance. Furthermore, the relatively high r value obtained is also an indicative of the closeness of irradiance and percent occurrence. The high percent occurrence of Neosiphonia seemed to occur with the incidence of highest irradiance at the surface of the water. However, a negative but strong correlation between salinity and percent occurrence of Neosiphonia was determined; this indicated that at high salinities, there was a lower percent occurrence of the epiphyte Neosiphonia. Temperature and turbidity showed very weak correlations with the percent Neosiphonia. Among the four environmental parameters measured and analyzed in the present study, irradiance and salinity showed the most positive and strongest correlations with the occurrence of Neosiphonia infections.

The results of the present study suggest that both varieties of K. alvarezii are best grown at the surface of the water for a period required to obtain the highest growth rate. However, the materials should be lowered to a depth range of 50–100 cm in order to benefit from the least impact of Neosiphonia. Furthermore, results showed that dipping of seedlings in AMPEP as prescribed here, prior to out-planting, was efficient for both improving the daily growth rate and increased productivity of both varieties of Kappaphycus and could be used as part of a management protocol to control or reduce the impact of Neosiphonia infection in commercial cultivation areas in the sea.