Seasonal occurrence and feeding habits of black marlin, <Emphasis Type="Italic">Istiompax indica</Emphasis>, around Yonaguni Island,southwestern Japan

The seasonal occurrence of black marlin, Istiompax indica, around Yonaguni Island, southwestern Japan, was analyzed. Black marlin was abundant from April to July, and the majority of landed fish were female (97%). None of these females was reproductively mature. The condition factor of females increased from April to July. Of 56 black marlin examined, 21 individuals contained 12 prey species; the most important prey species was skipjack tuna, Katsuwonus pelamis, and one neonatal silky shark, Carcharhinus falciformis, was also found. These results indicate that the occurrence of black marlin off Yonaguni Island was associated with feeding and not with spawning.     

Dynamic habitat suitability modelling reveals rapid poleward distribution shift in a mobile apex predator

Many taxa are undergoing distribution shifts in response to anthropogenic climate change. However, detecting a climate signal in mobile species is difficult due to their wide‐ranging, patchy distributions, often driven by natural climate variability. For example, difficulties associated with assessing pelagic fish distributions have rendered fisheries management ill‐equipped to adapt to the challenges posed by climate change, leaving pelagic species and ecosystems vulnerable. Here, we demonstrate the value of citizen science data for modelling the dynamic habitat suitability of a mobile pelagic predator (black marlin, Istiompax indica) within the south‐west Pacific Ocean. The extensive spatial and temporal coverage of our occurrence data set (n = 18 717), collected at high resolution (~1.85 km²), enabled identification of suitable habitat at monthly time steps over a 16‐year period (1998–2013). We identified considerable monthly, seasonal and interannual variability in the extent and distribution of suitable habitat, predominately driven by chlorophyll a and sea surface height. Interannual variability correlated with El Nino Southern Oscillation (ENSO) events, with suitable habitat extending up to ~300 km further south during La Nina events. Despite the strong influence of ENSO, our model revealed a rapid poleward shift in the geometric mean of black marlin habitat, occurring at 88.2 km decade^?1. By incorporating multiple environmental factors at monthly time steps, we were able to demonstrate a rapid distribution shift in a mobile pelagic species. Our findings suggest that the rapid velocity of climate change in the south‐west Pacific Ocean is likely affecting mobile pelagic species, indicating that they may be more vulnerable to climate change than previously thought.     

Movement ecology of black marlin Istiompax indica in the Western Indian Ocean

The black marlin Istiompax indica is an apex marine predator and is susceptible to overfishing. The movement ecology of the species remains poorly known, particularly within the Indian Ocean, which has hampered assessment of their conservation status and fisheries management requirements. Here, we used pop-up archival satellite tags to track I. indica movement and examine their dispersal. Forty-nine tags were deployed off Kenya during both the north-east (November–April) and south-west (August–September) monsoon seasons, providing locations from every month of the year. Individual I. indica were highly mobile and track distance correlated with the duration of tag attachment. Mean track duration was 38 days and mean track distance was >1800 km. Individuals dispersed in several directions: north-east into Somalian waters and up to northern Oman, east towards the Seychelles, and south into the Mozambique Channel. Their core habitat shifted seasonally and overlapped with areas of high productivity off Kenya, Somalia and Oman during the first half of the year. A second annual aggregation off the Kenyan coast, during August and September, did not coincide with high chlorophyll-a (chl-a) concentrations or thermal fronts, and the drivers of the species' presence and movement from this second aggregation was unclear. We tested their habitat preferences by comparing environmental conditions at track locations to the conditions at locations along simulated tracks based on the empirical data. Observed I. indica preferred cooler water with higher chl-a concentrations and stayed closer to the coast than simulated tracks. The rapid and extensive dispersal of I. indica from Kenya suggests that there is likely a single stock in the Western Indian Ocean, with individuals swimming between areas of high commercial catches off northern Somalia and Oman, and artisanal and recreational fisheries catches throughout East Africa and Mozambique.     

The Novel Application of Non-Lethal Citizen Science Tissue Sampling in Recreational Fisheries

Increasing fishing pressure and uncertainty surrounding recreational fishing catch and effort data promoted the development of alternative methods for conducting fisheries research. A pilot investigation was undertaken to engage the Australian game fishing community and promote the non-lethal collection of tissue samples from the black marlin Istiompax indica, a valuable recreational-only species in Australian waters, for the purpose of future genetic research. Recruitment of recreational anglers was achieved by publicizing the project in magazines, local newspapers, social media, blogs, websites and direct communication workshops at game fishing tournaments. The Game Fishing Association of Australia and the Queensland Game Fishing Association were also engaged to advertise the project and recruit participants with a focus on those anglers already involved in the tag-and-release of marlin. Participants of the program took small tissue samples using non-lethal methods which were stored for future genetic analysis. The program resulted in 165 samples from 49 participants across the known distribution of I. indica within Australian waters which was a sufficient number to facilitate a downstream population genetic analysis. The project demonstrated the potential for the development of citizen science sampling programs to collect tissue samples using non-lethal methods in order to achieve targeted research objects in recreationally caught species.     

Sex ratio and reproductive condition of four istiophorid billfishes in tropical regions of the eastern North Pacific Ocean: with special reference to striped marlin Kajikia audax (Philippi, 1887)

Sexual differences in the distribution pattern, sex ratio and the gonad condition of four istiophorid billfishes were investigated in three different tropical areas of the eastern North Pacific Ocean from September to November 2004. Sex ratios of striped marlin Kajikia audax were equal (female : male = 11 : 11) in the open‐ocean area (16–18°N, 118–134°W), biased to males (32 : 62) in the near‐continental area (13–16°N, 103–107°W), and biased to females (19 : 6) in the near‐equatorial area (5°N, 104–120°W). Sex ratios of sailfish Istiophorus platypterus in the near‐continental area and shortbill spearfish Tetrapturus angustirostris in the open‐ocean area were not biased from 1 : 1 (43 : 36, 11 : 13), and that of blue marlin Makaira nigricans in the near‐equatorial area was biased to females (14 : 1). Reproductively active females were found for striped marlin, sailfish and shortbill spearfish. Striped marlin showed a specific spawning area (the near‐continental area), and sexual dimorphism in its temporal distribution pattern, which was presumably related to reproduction. On the other hand, the limited distribution with evidence from spawning of sailfish and shortbill spearfish imply that these species complete their life history within limited areas.     

The rostral micro-tooth morphology of blue marlin,Makaira nigricans

Billfish rostra potentially have several functions; however, their role in feeding is unequivocal in some species. Recent work linked morphological variation in rostral micro-teeth to differences in feeding behavior in two billfish species, the striped marlin (Kajikia audax) and the sailfish (Istiophorus platypterus). Here, we present the rostral micro-tooth morphology for a third billfish species, the blue marlin (Makaira nigricans), for which the use of the rostrum in feeding behavior is still undocumented from systematic observations in the wild. We measured the micro-teeth on rostrum tips of blue marlin, striped marlin, and sailfish using a micro–computed tomography approach and compared the tooth morphology among the three species. This was done after an analysis of video-recorded hunting behavior of striped marlin and sailfish revealed that both species strike prey predominantly with the first third of the rostrum, which provided the justification to focus our analysis on the rostrum tips. In blue marlin, intact micro-teeth were longer compared to striped marlin but not to sailfish. Blue marlin had a higher fraction of broken teeth than both striped marlin and sailfish, and broken teeth were distributed more evenly on the rostrum. Micro-tooth regrowth was equally low in both marlin species but higher in sailfish. Based on the differences and similarities in the micro-tooth morphology between the billfish species, we discuss potential feeding-related rostrum use in blue marlin. We put forward the hypothesis that blue marlin might use their rostra in high-speed dashes as observed in striped marlin, rather than in the high-precision rostral strikes described for sailfish, possibly focusing on larger prey organisms.     

Habitat and food partitioning of billfishes (Xiphioidei)

Species composition and food habits of four istiophorid billfishes were investigated and compared in three different tropical areas of the eastern North Pacific Ocean by longline operations from September to November 2004. Sailfish Istiophorus platypterus, shortbill spearfish Tetrapturus angustirostris and blue marlin Makaira nigricans had specific habitat preferences and mainly occurred in the near‐continent area (13–16° N; 103–107° W), the open‐ocean area (16–18° N; 118–134° W) and the near‐equator area (5° N; 104–120° W). Small (<140 cm in lower jaw–fork length) striped marlin Kajikia audax mainly occurred in the near‐continent area; however, large (≥140 cm) individuals occurred throughout all three areas. Prey compositions of large K. audax in the three areas were different from one other reflecting the prey availability in each area. In the open‐ocean area, molid fishes were dominant in mass for both large K. audax (49%) and T. angustirostris (73%), and large K. audax also fed on ostraciid (33%) and scombrid fishes (15%). In the near‐continent area, tetraodontid fishes were dominant for large and small I. platypterus (54, 57%), and both large and small K. audax also fed on tetraodontid fishes (3, 12%). Large K. audax in this area fed mainly on scombrid fishes (86%). These results indicate that large K. audax show overlaps but little segregations of its prey with other billfishes. In the near‐equator area, stomach contents of large K. audax and M. nigricans were few and billfish prey items were thought to be scarce.     

Impact of Piriformospora indica on tomato growth and on interaction with fungal and viral pathogens

Piriformospora indica is a root endophytic fungus with plant-promoting properties in numerous plant species and induces resistance against root and shoot pathogens in barley, wheat, and Arabidopsis. A study over several years showed that the endophyte P. indica colonised the roots of the most consumed vegetable crop tomato. P. indica improved the growth of tomato resulting in increased biomass of leaves by up to 20%. Limitation of disease severity caused by Verticillium dahliae by more than 30% was observed on tomato plants colonised by the endophyte. Further experiments were carried out in hydroponic cultures which are commonly used for the indoor production of tomatoes in central Europe. After adaptation of inoculation techniques (inoculum density, plant stage), it was shown that P. indica influences the concentration of Pepino mosaic virus in tomato shoots. The outcome of the interaction seems to be affected by light intensity. Most importantly, the endophyte increases tomato fruit biomass in hydroponic culture concerning fresh weight (up to 100%) and dry matter content (up to 20%). Hence, P. indica represents a suitable growth promoting endophyte for tomato which can be applied in production systems of this important vegetable plant not only in soil, but also in hydroponic cultures.     

Direct sequencing of mitochondrial DNA detects highly divergent haplotypes in blue marlin (Makaira nigricans).

We were able to differentiate between species of billfish (Istiophoridae family) and to detect considerable intraspecific variation in the blue marlin (Makaira nigricans) by directly sequencing a polymerase chain reaction (PCR)-amplified, 612-bp fragment of the mitochondrial cytochrome b gene. Thirteen variable nucleotide sites separated blue marlin (n = 26) into 7 genotypes. On average, these genotypes differed by 5.7 base substitutions. A smaller sample of swordfish from an equally broad geographic distribution displayed relatively little intraspecific variation, with an average of 1.3 substitutions separating different genotypes. A cladistic analysis of blue marlin cytochrome b variants indicates two major divergent evolutionary lines within the species. The frequencies of these two major evolutionary lines differ significantly between Atlantic and Pacific ocean basins. This finding is important given that the Atlantic stocks of blue marlin are considered endangered. Migration from the Pacific can help replenish the numbers of blue marlin in the Atlantic, but the loss of certain mitochondrial DNA haplotypes in the Atlantic due to overfishing probably could not be remedied by an influx of Pacific fish because of their absence in the Pacific population. Fishery management strategies should attempt to preserve the genetic diversity within the species. The detection of DNA sequence polymorphism indicates the utility of PCR technology in pelagic fishery genetics.     

GEOGRAPHIC DISTRIBUTION OF MOLECULAR VARIANCE WITHIN THE BLUE MARLIN (MAKAIRA NIGRICANS): A HIERARCHICAL ANALYSIS OF ALLOZYME,SINGLE-COPY NUCLEAR DNA,AND MITOCHONDRIAL DNA MARKERS

This study presents a comparative hierarchical analysis of variance applied to three classes of molecular markers within the blue marlin (Makaira nigricans). Results are reported from analyses of four polymorphic allozyme loci, four polymorphic anonymously chosen single-copy nuclear DNA (scnDNA) loci, and previously reported restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA). Samples were collected within and among the Atlantic and Pacific Oceans over a period of several years. Although moderate levels of genetic variation were detected at both polymorphic allozyme (H = 0.30) and scnDNA loci (H = 0.37), mtDNA markers were much more diverse (h = 0.85). Allele frequencies were significantly different between Atlantic and Pacific Ocean samples at three of four allozyme loci and three of four scnDNA loci. Estimates of allozyme genetic differentiation (θ_O) ranged from 0.00 to 0.15, with a mean of 0.08. The θ_O values for scnDNA loci were similar to those of allozymes, ranging from 0.00 to 0.12 with a mean of 0.09. MtDNA RFLP divergence between oceans (θ_O = 0.39) was significantly greater than divergence detected at nuclear loci (95% nuclear confidence interval = 0.04–0.11). The fourfold smaller effective population size of mtDNA and male-mediated gene flow may account for the difference observed between nuclear and mitochondrial divergence estimates.     

Reproductive biology and spatiotemporal patterns of spawning in striped marlin Kajikia audax

This study presents the first histology‐based assessment of the reproductive dynamics of south‐west Pacific striped marlin Kajikia audax. Maturity and reproductive status were assessed from histological sections of ovaries (n = 234) and testes (n = 243) of fish caught in commercial longline and recreational fisheries between 2006 and 2009. Spawning peaked in the Coral Sea during November and December at sea surface temperatures between 24·8 and 28·3° C. Lower jaw fork length (L_LJF) at 50% maturity (L_LJF50), a key variable for stock assessment, was estimated to be 2100 ± 102 mm (mean + s.e .) for females and 1668 ± 18 mm for males. Unlike large pelagic tunas Thunnus spp., the proportion of females increased with length and spawning fish formed multiple large‐scale aggregations within a broad latitudinal band. This study provides a starting point for biological parameters needed for stock assessment and conservation of K. audax and introduces the multiple aggregation spawning concept as a reproductive mechanism to explain genetic heterogeneity observed in some highly migratory species.     

Reproductive biology of the white marlin (Kajikia albida) in the southwestern and equatorial Atlantic Ocean

The present work describes the reproductive biology of the white marlin (Kajikia albida) caught in the southwestern and equatorial Atlantic Ocean. The gonads of 924 fish were collected by observers on board Brazilian tuna longliners, between November 2004 and December 2006. The spawning season was assessed by the monthly frequency distribution of distinct stages of maturity and monthly mean female gonadosomatic index GSI. Sixty-one percent (n = 656) of the fish examined were female, with a Lower Jaw Fork Length (LJFL) between 83 and 236 cm (mean = 155.5 ± 16.63). The 268 males had a LJFL between 90 and 220 cm (mean = 152.3 ± 34.62). Although the northeastern region of Brazil does not appear to be a significant spawning area for the species, the results suggest a higher reproductive activity in the third quarter of the year. Using a Bayesian logistic model approach, length at 50% maturity was estimated at 145.04 cm (credibility interval of 95%, 143.94–146.09 cm), for females, and at 140.03 cm (credibility interval of 95%, 137.28–142.52 cm), for males.     

Diet composition,feeding niche partitioning and trophic organisation of large pelagic predatory fishes in the eastern Arabian Sea

Information on the ecology and feeding behaviour of the large oceanic predatory fishes is crucial for the ecosystem approaches to fisheries management models. Co-existing large pelagic predators in the open oceans may avoid competition for the limited forage by resource partitioning on spatial, temporal or trophic levels. To test this, we studied the prey species composition, diet overlap, trophic level, and trophic organisation of 12 large predatory fishes co-existing in the eastern Arabian Sea. Stomach contents of 1,518 specimens caught by exploratory longline operations in the Indian Exclusive Economic Zone during the years 2006–2009 were analysed. Finfishes were dominant prey of all species except blue marlin (Makaira nigricans) and yellowfin tuna (Thunnus albacares), which fed mainly on cephalopods, and long-snouted lancetfish (Alepisaurus ferox) and pelagic stingray (Pteroplatytrygon violacea), which fed mainly on crustaceans. Common dolphinfish (Coryphaena hippurus) and yellowfin tuna fed on a wider variety of prey than the other species, while the diets of lancetfish and black marlin (Istiompax indica) were narrowest. Pelagic stingray and great barracuda (Sphyraena barracuda) fed on species occupying epipelagic waters, whereas the contribution of mesopelagic prey was higher in the diets of swordfish (Xiphias gladius) and pelagic thresher (Alopias pelagicus). Trophic levels of these fishes ranged from 4.13 to 4.37. Diet overlap index revealed that some of the large pelagic predatory fishes share common prey species. Cluster analysis of the diets revealed four distinct trophic guilds namely ‘flyingfish feeders’ (common dolphinfish and great barracuda); ‘mesopelagic predators’ (pelagic thresher and swordfish); ‘crab feeders’ (lancetfish, pelagic stingray and silky shark) and ‘squid feeders’ (yellowfin tuna, Indo-Pacific sailfish (Istiophorus platypterus), skipjack tuna (Katsuwonus pelamis), black marlin and blue marlin). Large predatory fishes of the eastern Arabian Sea target different prey types, and limit their vertical extent and time of feeding to avoid competing for prey.     

Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between <Emphasis Type="Italic">japonica</Emphasis> and <Emphasis Type="Italic">indica</Emphasis> rice varieties

Four amino acids were variable between the ‘active’ indica-type and ‘inactive’ japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the ‘active’ indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B₁ chains by elongating short A and B₁ chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.     

Selecting genetic transformants of indica and indica-derived rice cultivars using bispyribac sodium and a mutated ALS gene

Bispyribac sodium (BS), a pyrimidinyl carboxy herbicide, is a well-known inhibitor of acetolactate synthase (ALS) activity. ALS is an enzyme in the biosynthetic pathway for branched-chain amino acids. A mutant form of rice ALS (OsmALS [W548L/S627I]) that confers resistance to BS can be used as an in vitro selection marker gene for plant transformation. Since indica and indica-derived cultivars are thought to have lower BS sensitivity than japonica rice, the application of BS as a selectable reagent for genetic transformation in indica and indica-derived cultivars is more challenging than for japonica cultivars. In this study, callus and seedlings of eight different rice cultivars (five indica-derived cultivars, two indica cultivars and one japonica cultivar) were tested for BS sensitivity. Our study indicates for the first time that callus shows a higher sensitivity to BS than seedlings in indica and indica-derived cultivars. We used BS with OsmALS [W548L/S627I] to select transformed calli, and transgenic rice plants from indica and indica-derived cultivars were successfully obtained.     

Biochemical analysis of the allergenic pollen ofMadhuca indica Gmel. andSpathodea campanulata Beauv

Summary The present paper deals with the biochemical analysis in the form of quantitative estimation of carbohydrates, proteins, nucleic acids, minerals, ash and moisture of the pollen of two allergenic plants, i.e.Manhuca indica andSpathodea campanulata. Carbohydrates constitute the major dry matter fractions in both the pollen types with highest amount inM. indica (25.37%). Soluble proteins were estimated along with lipid content.S. campanulata showed 12.74% protein andM. indica 11.58%. The estimation of nucleic acids revealed the presence of DNA from 0.36 to 0.72% and RNA from 0.28 to 1.03%. Minerals were found in very low amounts ranging from 0.14 to 0.19% for Calcium and 0.15 to 0.20% for Magnesium. The ash and moisture content were also found variable. The present findings revealed that these chemical constituents may be responsible for the control of the rate and type of allergic reaction caused by the pollen of these two plants.     

Effects of post-application irrigation and substrate moisture on the efficacy of entomopathogenic nematodes against western flower thrips, Frankliniella occidentalis

The efficacy of entomomatogenic nematodes (Steinernema bicornutum Tallosi, Peters and Ehlers and/or Heterorhabditis indica LN2 Poinar, Karunakar and David) against the soil‐dwelling life stages of western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) was assessed under different moisture conditions in a commercial plant‐growing substrate in laboratory experiments. In the first experiment, both nematode species were tested at substrate moisture ranges of 67, 78, 88, or 95% relative moisture content, that were maintained before applying the nematodes at 100 or 400 infective juveniles (IJs) cm^?2. In the second experiment, 10, 25, 50, 100, or 120 ml irrigation water, resulting in relative moisture contents of 72, 81, 90, 99%, or more than the saturation level of the substrate, respectively, was applied to the substrate. Heterorhabditis indica LN2 was applied either in 3 ml water and followed by irrigation, or by suspending the infective juveniles in the water amounts indicated above to apply the nematodes in higher volume. Results indicated that at the higher application rate, initial moisture content did not significantly affect the efficacy of H. indica LN2. On the other hand, increasing moisture content resulted in an improved efficacy of H. indica LN2 and S. bicornutum at lower and higher application rates, respectively. Similar thrips control levels of 44 and 60% at the lower and higher application rate of H. indica LN2, respectively, were obtained at 88% relative moisture content. In the second experiment, higher and statistically similar thrips mortality of 40 and 50% at lower and higher application rates of H. indica LN2, respectively, were obtained when the infective juveniles were applied in a high volume suspension of 100 ml, or when followed by irrigation with 25 ml water, resulting in both cases in 81% relative moisture content. Generally, efficacies of the nematodes for thrips control can be improved by using an appropriate moisture content and/or post‐application irrigation. Thus, the high nematode application rates required for successful F. occidentalis control can be partly attributed to substrate moisture content and/or post‐application irrigation.     

Rapid identification of the north-western Pacific billfish species using quantitative real-time polymerase chain reaction techniques

Billfishes are important fishery resources traded and consumed worldwide. As morphological traits are usually removed during processing, molecular methods are applied to identify billfish products. In this study, the approaches of quantitative real-time PCR were developed to identify the six billfish species (Istiompax indica, Istiophorus platypterus, Kajikia audax, Makaira nigricans, Tetrapturus angustirostris and Xiphias gladius) widely distributed in the north-western Pacific Ocean. The developed singleplex systems showed high fidelities to each of the six species via either examining the ΔCt values or melting curve patterns. For samples containing multiple species, individual species are identifiable by a quantitative real-time PCR assay that includes all the singleplex systems. A multiplex system was also developed to identify unknown samples composed of a single species. The methods developed in this study provide a fast and high-throughput manner to identify the north-western Pacific billfish species when morphological traits are unavailable, such as in processed products.     

Compatibility of <Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> as dual inoculants in black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

The compatibility of two biological inoculants, Trichoderma harzianum, a mycoparasitic biological control fungus and Piriformospora indica, a root colonizing plant-growth promoting endophytic fungus was evaluated using tissue cultured black pepper plantlets. We report, for the first time, the ability of P. indica to colonize black pepper, a perennial climber. T. harzianum inhibited the growth of P. indica in an in vitro dual culture plate assay. Simultaneous inoculation with both biological inoculants of tissue cultured black pepper plantlets negatively influenced root colonization by P. indica. However, when P. indica was applied initially followed 30 days later by T. harzianum, there was increased root colonization by the root endophyte P. indica and beneficial effects were found on the growth of the black pepper plants. The present study also showed that the efficacy of inoculation of the two fungal biological agents can be increased by sequential application of P. indica at the hardening stage followed by T. harzianum during transplanting into a soil-sand mixture.